Combination Therapy of Acellular Pro-Tolerogenic and Pro-Inflammatory Preparations for Modulating the Immune System

ABSTRACT

This disclosure relates to acellular-based therapies for modulating the level of regulatory T cells (Treg) and/or the level of pro-inflammatory T cells (Th17/Th1). To provide these therapeutic effects, a combination comprising at least one acellular pro-tolerogenic preparation and at least one acellular pro-inflammatory preparation are administered sequentially.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/325,265 filed 10 Jan. 2017, which is a national phase applicationunder 35 U.S.C. § 371 of International Application No. PCT/CA2015/050647filed 10 Jul. 2015, which claims priority to U.S. ProvisionalApplication No. 62/023,072 filed 10 Jul. 2014. The entire contents ofeach of the above-referenced disclosures is specifically incorporated byreference herein without disclaimer.

TECHNOLOGICAL FIELD

The present disclosure relates to the use of a combination ofacellular-based preparations (obtained from contacting allogeneicleukocytes) to modulate the ratio in the level of regulatory T (Treg)cells to the level of pro-inflammatory T cells. The combination can beused to temporarily modulate the Treg cells/pro-inflammatory T cellsratio or adjust the ratio in view of the progression of the conditionthat is being prevented, treated and/or alleviated.

BACKGROUND

The immune response evolved to be inherently adaptable depending on thechallenges it meets. In some instances, pregnancy for example, theimmune response is reduced and tolerates immunological triggers orinsults. In other instances, a microbial infection for example, theimmune response is strong and allows the return to an homeostatic state.

However, in some individuals the immune balance is pathologically tippedeither towards inflammation (e.g., a pro-inflammatory state, as observedin autoimmune diseases for example) or anergy (e.g., a pro-tolerogenicstate, as observed in proliferation associated disorders for example)which leads to the onset of various conditions which may be long-livedand detrimental to the afflicted individuals. In order to mitigate suchconditions, various therapeutics have been designed to restore an immunebalance which will limit or prevent the pathological consequencesassociated with the onset of such conditions.

Therapeutics that are capable of restoring the immune balance, and morespecifically capable of modulating the ratio of T regulatory (Treg)cells to pro-inflammatory T cells, have been described. A first class ofbiological therapeutics has been designed to decrease the ratio ofTreg/pro-inflammatory cells in order to intentionally induce a moreinflammatory immune state in individuals having a low or inappropriateimmune response (refer to, for example, PCT patent applicationsPCT/CA2013/050546 (published under WO2014/008611) andPCT/CA2013/050963). These biological therapeutics are especially usefulin mediating therapeutic benefits in individuals afflicted by aproliferation-associated disorder such as cancer. Other biologicaltherapeutics have been designed to increase the ratio ofTreg/pro-inflammatory T cells to intentionally induce a more tolerogenicimmune state in individuals having an exacerbated immune response (referto, for example, U.S. patent application Ser. No. 13/941,303 (publishedunder US 2014/0017218), PCT patent applications PCT/CA2013/050547(published under WO2014/008612), PCT/CA2013/050543 (published underWO2014/008608), PCT/CA2013/050544 (published under WO2014/008609) andPCT/CA2013/050545 (published under WO2014/008610)). This second class ofbiological therapeutics can provide therapeutic benefits in individualsafflicted by an auto-immune disease or at risk of rejecting a graft.

Even though some of the biological therapeutics have been show to inducelong-lived beneficial immune modulating effects in individuals havingreceived them, there exists a need for further modulating the immuneresponse in some situations. For example, while providing therapeuticbenefits to individuals afflicted by an auto-immune disease (byintentionally inducing a pro-tolerogenic immune state), these biologicaltherapeutics also impede the immune response of the treated individualstowards a vaccine. As such, it may be beneficial for individuals havingreceived biological therapeutics intentionally inducing apro-tolerogenic state to have the ability, at least temporarily, tomount a robust immune response against an immunogen, such as thecomponents of a vaccine.

In another example, individuals having received a biological therapeuticintentionally inducing a pro-inflammatory state to favor tumorresorption could also benefit from a more pro-tolerogenic state prior toa tissue or a cell graft. As such, it may be beneficial for individualshaving received biological therapeutics intentionally inducing apro-inflammatory state to have the ability, at least temporarily, toavoid mounting an immune response against a grafted tissue or atransplanted cell.

It would be highly desirable to be provided with therapeuticcombinations capable of modulating the immune response in an individualto provide immune stimulation when a pro-tolerogenic immune state wasintentionally induced or immune tolerance when a pro-inflammatory immunestate was intentionally induced. In some embodiments, it would also behighly desirable for some individuals to revert back to theintentionally induced pro-tolerogenic immune state or the intentionallyinduced pro-inflammatory immune state.

BRIEF SUMMARY

One aim of the present disclosure is to provide a therapeuticcombination of acellular-based preparations capable of inducing, in asequential manner, a state of immune stimulation and a state of immunetolerance. The acellular-based preparations and therapies presentedherewith are derived from the contact of at least two distinct leukocytepopulations which are considered allogeneic with respect to one another.The two leukocyte populations are contacted under conditions so as toallow either a pro-inflammatory allo-recognition and ultimately induceimmune stimulation or a pro-tolergenic allo-recognition to ultimatelyinduce immune tolerance. The two leukocyte populations can be contactedin vitro, ex vivo or in vivo to induce immune stimulation and/or apro-inflammatory state. The acellular-based preparations are obtained ina process which limits or inhibits the degradation of nucleic acids,such as miRNAs and can even include an mi-RNA enrichment step.

According to a first aspect, the present disclosure relates to atherapeutic combination comprising at least one acellularpro-tolerogenic preparation and at least one acellular pro-inflammatorypreparation. In such combination, the at least one acellularpro-tolerogenic preparation and the at least one acellularpro-inflammatory preparation are adapted for a sequential administrationto a subject. The at least one acellular pro-tolerogenic preparation isobtained by a first process comprising: (i) associating alow-immunogenic biocompatible polymer to a cytoplasmic membrane of afirst leukocyte to obtain a first modified leukocyte; (ii) contactingthe first modified leukocyte with a second leukocyte under conditions toallow a pro-tolerogenic allo-recognition to provide a pro-tolerogenicconditioned preparation, wherein the first modified leukocyte isallogeneic to the second leukocyte; (iii) removing the first modifiedleukocyte and the second leukocyte from the pro-tolerogenic conditionedpreparation under conditions to inhibit RNA degradation so as to obtaina pro-tolerogenic composition enriched in acellular pro-tolerogeniccomponents; and (iv) formulating the pro-tolerogenic composition of step(iii), under conditions to inhibit RNA degradation, in the acellularpro-tolerogenic preparation for administration to the subject. The atleast one pro-inflammatory preparation is obtained by a second processcomprising: (a) contacting a third leukocyte with a fourth leukocyteunder conditions to allow a pro-inflammatory allo-recognition to providea pro-inflammatory conditioned preparation, wherein the third leukocyteis allogeneic to the fourth leukocyte; (b) removing the third leukocyteand the fourth leukocyte from the pro-inflammatory conditionedpreparation under conditions to inhibit RNA degradation so as to obtaina pro-inflammatory composition enriched in acellular pro-inflammatorycomponents; and (c) formulating the pro-inflammatory composition of step(b), under conditions to inhibit RNA degradation, in the acellularpro-inflammatory preparation for administration to the subject. In anembodiment, the acellular pro-tolerogenic preparation is adapted to beadministered to the subject prior to the acellular pro-inflammatorypreparation. In still another embodiment, the therapeutic combinationfurther comprises at least two acellular pro-tolerogenic preparations,wherein the first acellular pro-tolerogenic preparation is adapted to beadministered to the subject prior to the acellular pro-inflammatorypreparation and wherein the second acellular pro-tolerogenic preparationis adapted to be administered to the subject after the acellularpro-inflammatory preparation. In yet another embodiment, the acellularpro-tolerogenic preparation is adapted to be administered to the subjectafter the acellular pro-inflammatory preparation. In another embodiment,the therapeutic combination further comprises at least two acellularpro-inflammatory preparations, wherein the first acellularpro-inflammatory preparation is adapted to be administered to thesubject prior to the acellular pro-tolerogenic preparation and whereinthe second acellular pro-inflammatory preparation is adapted to beadministered to the subject after the acellular pro-tolerogenicpreparation.

In still another embodiment, the first process, at step (i), furthercomprises covalently binding the low-immunogenic biocompatible polymerto a membrane-associated protein of the cytoplasmic membrane of thefirst leukocyte. In still a further embodiment, the low-immunogenicbiocompatible polymer is a polyethylene glycol (PEG), such as, forexample a methoxy polyethylene glycol (mPEG). In a further embodiment,the first process further comprises covalently binding the mPEG bycontacting the first leukocyte with methoxypoly(-ethylene glycol)succinimidyl valerate. In yet another embodiment, step (ii) of the firstprocess occurs in vitro. In such embodiment, the first process, at step(ii), can further comprise culturing the first modified leukocyte andthe second leukocyte. In an embodiment, the pro-tolerogenic conditionedpreparation is a supernatant of the cell culture. In another embodiment,the first process, prior to step (ii), further comprises preventing oneof the first modified leukocyte or the second leukocyte fromproliferating. In still a further embodiment, step (ii) of the firstprocess occurs in vivo. In such embodiment, the first process, at step(ii), can further comprise administering the first modified leukocyte toa mammal having the second leukocyte. In yet another embodiment, thepro-tolerogenic conditioned preparation is a plasma of the mammal. Inanother embodiment, the first process, prior to step (ii), furthercomprises preventing the first modified leukocyte from proliferatingprior to administration to the mammal. In yet a further embodiment, thefirst process, at step (iii), further comprises removing componentshaving an average molecular weight of more than about 10 kDa from thepro-tolerogenic conditioned preparation, for example, the first process,at step (iii), can further comprise filtering out components having theaverage molecular weight of more than about 10 kDa from thepro-tolerogenic conditioned preparation. In still another embodiment,the first process, at step (iii), further comprises enriching thepro-tolerogenic conditioned preparation in at least one miRNA species.In another embodiment, the first process, at step (iv), furthercomprises formulating the acellular pro-tolerogenic composition forintravenous administration to the subject.

In yet another embodiment, step (a) of the second process occurs invitro. In such embodiment, the second process, at step (a), can furthercomprise culturing the third leukocyte and the fourth leukocyte. In anembodiment, the pro-inflammatory conditioned preparation is asupernatant of the cell culture. In still a further embodiment, thesecond process, prior to step (a), further comprises preventing one ofthe third leukocyte or the fourth leukocyte from proliferating. Inanother embodiment, step (a) of the second process occurs in vivo. Insuch embodiment, the second process can further comprise administeringthe third leukocyte to a mammal having the fourth leukocyte. In anembodiment, the pro-inflammatory conditioned preparation is a plasmafrom the mammal. In still another embodiment, the second process, priorto step (a), further comprises preventing the third leukocyte fromproliferating prior to administration to the mammal. In yet anotherembodiment, the second process, at step (b), further comprises removingcomponents having an average molecular weight of more than about 10 kDafrom the pro-inflammatory conditioned preparation, for example, thesecond process, at step (b), can further comprise filtering outcomponents having the average molecular weight of more than about 10 kDafrom the pro-inflammatory conditioned preparation. In an embodiment, thesecond process, at step (b), further comprises enriching thepro-inflammatory conditioned preparation in at least one miRNA species.In yet another embodiment, the second process, at step (c), furthercomprises formulating the acellular pro-inflammatory composition forintravenous administration to the subject.

In still another embodiment, at least one of the first leukocyte, thesecond leukocyte, the third leukocyte or the fourth leukocyte is a Tcell (a CD4-positive T cell or a CD8-positive T cell). In yet anotherembodiment, at least one of the first leukocyte, the second leukocyte,the third leukocyte and the fourth leukocyte is a peripheral bloodmononucleated cell. In a further embodiment, at least one of the firstleukocyte, the second leukocyte, the third leukocyte and the fourthleukocyte is a splenocyte.

In another embodiment, the acellular pro-tolerogenic preparation has atleast one miRNA species presented in FIG. 9, listed in any one of Tables1A to 1D, listed in any one of Tables 2A to 2D or presented in any oneof FIGS. 8A to 8C. In a further embodiment, the acellularpro-inflammatory preparation has at least one miRNA species presented inFIG. 9, listed in any one of Tables 1A to 1D, listed in any one ofTables 2A to 2D or presented in any one of FIGS. 8A to 8C.

In a second aspect, the present disclosure provides a therapeutic kitcomprising at least one acellular pro-tolerogenic preparation as definedherein, at least one acellular pro-inflammatory preparation as definedherein and instructions for using the at least one acellularpro-tolerogenic preparation and the acellular pro-inflammatorypreparation in a sequential manner. In an embodiment, the instructionsspecify that the acellular pro-tolerogenic preparation foradministration to the subject prior to the acellular pro-inflammatorypreparation. In still another embodiment, the therapeutic kit furthercomprises at least two acellular pro-tolerogenic preparations, whereinthe instructions specify that the first acellular pro-tolerogenicpreparation is for administration to the subject prior to the acellularpro-inflammatory preparation and the second acellular pro-tolerogenicpreparation is for administration to the subject after the acellularpro-inflammatory preparation. In still another embodiment, theinstructions specify that the acellular pro-tolerogenic preparation isfor administration to the subject after the acellular pro-inflammatorypreparation. In yet another embodiment, the therapeutic kit furthercomprises at least two acellular pro-inflammatory preparations, whereinthe instructions specify that the first acellular pro-inflammatorypreparation is for administration to the subject prior to the acellularpro-tolerogenic preparation and the second acellular pro-inflammatorypreparation is for administration to the subject after the acellularpro-tolerogenic preparation.

In a third aspect, the present disclosure provides a method ofmodulating a ratio of the level of regulatory T (Treg) cells to thelevel of pro-inflammatory T cells in a subject in need thereof, saidmethod comprising administering a therapeutic amount of at least oneacellular pro-inflammatory preparation as defined herein to the subjecthaving received a therapeutic amount of at least one acellularpro-tolerogenic preparation as defined herein. In an embodiment, themethod further comprises identifying that the subject is need of adecrease of the ratio of the level of Treg cells to the level ofpro-inflammatory T cells prior to the administration of the at least oneacellular pro-inflammatory preparation. In another embodiment, themethod further comprises administering to the subject a therapeuticamount of the at least one acellular pro-tolerogenic preparation priorto the administration of the therapeutic amount of the at least oneacellular pro-inflammatory preparation. In still another embodiment, themethod further comprises identifying that the subject is need of anincrease of the ratio of the level of Treg cells to the level ofpro-inflammatory T cells prior to the administration of the at least oneacellular pro-tolerogenic preparation. In an embodiment, the need forthe decrease of the ratio is for treating, preventing and/or alleviatingthe symptoms associated with a condition caused or exacerbated by areduced immune response in the subject. In still another embodiment, thecondition is a proliferation-associated disorder. In yet anotherembodiment, the proliferation-associated disorder is cancer. In afurther embodiment, the condition is an infection (for example, aparasitic infection, a viral infection, a bacterial infection and/or afungal infection). In still another embodiment, the condition is animmune response to a vaccine. In a further embodiment, the need for theincrease of the ratio is for treating, preventing and/or alleviating thesymptoms associated to an auto-immune disease afflicting the subject(such as, for example, type I diabetes, rheumatoid arthritis, multiplesclerosis, psoriasis, lupus, immune thrombocytopenia, experimentalautoimmune encephalomyelitis, autoimmune uveitis, inflammatory boweldisease, scleroderma and/or Crohn's disease). In yet another embodiment,the need for the increase of the ratio is for preventing or limiting therejection of transplanted cells or tissue in the subject. In still afurther embodiment, the transplanted cells or tissue are allogeneic orxenogeneic to the subject.

In a fourth aspect, the present disclosure provides a method ofmodulating a ratio of the level of regulatory T (Treg) cells to thelevel of pro-inflammatory T cells in a subject in need thereof, saidmethod comprising administering a therapeutic amount of at least oneacellular pro-tolerogenic preparation as defined herein to the subjecthaving received a therapeutic amount of at least one acellularpro-inflammatory preparation as defined herein. In an embodiment, themethod further comprises identifying that the subject is need of anincrease of the ratio of the level of Treg cells to the level ofpro-inflammatory T cells prior to the administration of the at least oneacellular pro-tolerogenic preparation. In still another embodiment, themethod further comprises administering to the subject a therapeuticamount of the at least one acellular pro-inflammatory preparation priorto the administration of the therapeutic amount of the at least oneacellular pro-tolerogenic preparation. In yet another embodiment, themethod further comprises identifying that the subject is need of adecrease of the ratio of the level of Treg cells to the level ofpro-inflammatory T cells prior to the administration of the at least oneacellular pro-inflammatory preparation. In an embodiment, the need forthe decrease of the ratio is for treating, preventing and/or alleviatingthe symptoms associated with a condition caused or exacerbated by areduced immune response in the subject. In still another embodiment, thecondition is a proliferation-associated disorder. In yet anotherembodiment, the proliferation-associated disorder is cancer. In afurther embodiment, the condition is an infection (for example, aparasitic infection, a viral infection, a bacterial infection and/or afungal infection). In still another embodiment, the condition is animmune response to a vaccine. In a further embodiment, the need for theincrease of the ratio is for treating, preventing and/or alleviating thesymptoms associated to an auto-immune disease afflicting the subject(such as, for example, type I diabetes, rheumatoid arthritis, multiplesclerosis, psoriasis, lupus, immune thrombocytopenia, experimentalautoimmune encephalomyelitis, autoimmune uveitis, inflammatory boweldisease, scleroderma and/or Crohn's disease). In yet another embodiment,the need for the increase of the ratio is for preventing or limiting therejection of transplanted cells or tissue in the subject. In still afurther embodiment, the transplanted cells or tissue are allogeneic orxenogeneic to the subject.

Throughout this text, various terms are used according to their plaindefinition in the art. However, for purposes of clarity, some specificterms are defined below.

Allogeneic cell. A cell is considered “allogeneic” with respect toanother cell if both cells are derived from the same animal species butpresents sequence variation in at least one genetic locus. A cell isconsidered “allogeneic” with respect to a subject if the cell is derivedfrom the same animal species as the subject but presents sequencevariation in at least one genetic locus when compared to the subject'srespective genetic locus. Allogeneic cells induce an immune reaction(such as a cell-based immune reaction, a rejection for example) whenthey are introduced into an immunocompetent host. In an embodiment, afirst cell is considered allogeneic with respect to a second cell if thefirst cell is HLA-disparate (or HLA-mismatched) with respect to thesecond cell.

Allo-recognition. As it is known in the art, the term “allo-recognition”(also spelled allorecognition) refers to an immune response to foreignantigens (also referred to as alloantigens) from members of the samespecies and is caused by the difference between products of highlypolymorphic genes. Among the most highly polymorphic genes are thoseencoding the MHC complex which are most highly expressed on leukocytesthough other polymorphic proteins may similarly result in immunerecognition. These polymorphic products are typically recognized by Tcells and other mononuclear leukocytes. In the context of the presentdisclosure, the term “pro-inflammatory allo-recognition” refers to animmune response associated with the expansion of pro-inflammatory Tcells and/or the differentiation of naïve T cells into pro-inflammatoryT cells. Pro-inflammatory allo-recognition in vivo mediates cell ortissue injury and/or death and loss of cell or tissue function. Still inthe context of the present disclosure, the term “pro-tolerogenicallo-recognition” refers to an immune response associated with theexpansion of Treg cells and/or the differentiation of naïve T cells intoTreg cells and/or a decrease in the expansion of pro-inflammatory Tcells (e.g., Th1, Th17 cells) and/or differentiation of naïve T cells topro-inflammatory T cells. A pro-tolerogenic allo-recognition is usuallyconsidered weaker than a pro-inflammatory allo-recognition. Further, anin vivo pro-tolerogenic allo-recognition does not lead to significantcell or tissue injury and/or death nor to loss of cell or tissuefunction.

Anergy and Tolerance. In the present context, the term “anergy” refersto a non-specific state of immune unresponsiveness to an antigen towhich the host was previously sensitized to or unsensitized to. It canbe characterized by a decrease or even an absence of lymphokinesecretion by viable T cells when the T cell receptor is engaged by anantigen. In the present context, the term “tolerance” (also referred toas a pro-tolerogenic state) refers to an acquired specific failure ofthe immunological mechanism to respond to a given antigen, induced byexposure to the antigen (e.g., a tumor antigen for example). Tolerancerefers to a specific nonreactivity of the immune system to a particularantigen, which is capable, under other conditions, of inducing an immuneresponse. However, in the present context, the terms “anergy” and“tolerance” are used interchangeably since the compositions and methodspresented herewith can be used to achieve both anergy and tolerance.

Autologous cell. A cell is considered “autologous” with respect toanother cell if both cells are derived from the same individual or fromgenetically identical twins. A cell is considered “autologous” to asubject, if the cell is derived from the subject or a geneticallyidentical twin. Autologous cells do not induce an immune reaction (suchas a rejection) when they are introduced into an immunocompetent host.

Conditions associated with a reduced (low or inappropriate) immuneresponse. In the context of the present disclosure, the subjectsafflicted by these conditions have increased ratio of Treg topro-inflammatory T cells when compare to the same ratio of sex- andage-matched healthy subjects. Alternatively, the subjects afflicted bythese conditions may have normal ratios of Treg to pro-inflammatory Tcells but exhibit a reduced to absent proinflammatory response toantigenic stimuli. In some embodiments, the immune system of subjectsafflicted by a condition associated with a low, repressed orinappropriate immune response is in a state of anergy. The immune systemof some of the subjects afflicted by these conditions fails to producetarget specific pro-inflammatory cell (T and B lymphocytes) capable ofrecognizing and destroying abnormal cells (e.g., cancer cells orinfected cells). Alternatively, the immune system of some of thesubjects afflicted by these conditions exhibit elevated levels ofregulatory T and B cells that inhibit normal pro-inflammatory T and Bcells from exerting their function (i.e. inducing a partial or completeimmune suppression) thereby preventing destruction of an abnormal cellof cell aggregates. One of these conditions is aproliferation-associated disorder (such as, for example, cancer).Another of these conditions is an infection (such as for example aparasitic infection).

Immune stimulation. In the present context, the term “immunestimulation” or “pro-inflammatory state” refers to a state of immuneresponsiveness to an antigen and such response is independent of thehost's previous sensitization to the antigen. It can be characterized byan increase or a modulation in the level of lymphokine secretion byviable T cells when the T cell receptor is engaged by an antigen. In thepresent context, the term “stimulation” refers to an acquired specificactivation of the immunological mechanism to respond to a given antigen,induced by exposure to the antigen. In the context of the presentdisclosure, the immune stimulation is considered therapeutic andspecifically excludes inflammatory diseases, conditions and/ordisorders.

Immunogenic cell. A first cell is considered immunogenic with respect toa second cell when it is able to induce an immune response in the lattercell. In some embodiment, the immune response is in vitro (e.g., a mixedlymphocyte reaction) or can be observed in vivo (e.g., in a subjecthaving the second cell and having received the first cell). The secondcell can be located in an immunocompetent subject. Preferably, theimmune response is a cell-based immune response in which cellularmediator can be produced. In the context of the present disclosure, theimmunogenic cells are immune cells, such as white blood cells orleukocytes.

Immunogenic cell culture conditions. A cell culture is considered to beconducted in immunogenic conditions when it allows the establishment ofa pro-inflammatory immune response between two distinct and unmodifiedleukocytes (and, in an embodiment, when it allows allo-recognition).Preferably, the pro-inflammatory immune response is a cell-based immuneresponse in which cellular mediator can be produced. For example, thecell culture conditions can be those of a mixed lymphocyte reaction(primary or secondary).

Infection. As used in the context of the present disclosure, the term“infection” or “infective disease” is a condition caused by the presenceand proliferation of an infectious agent which induces a state of low orrepressed immune response (e.g., anergy). In some embodiments, theinfection is caused by a parasite and in such instance, it is referredto as a “parasitic” infection. There are mainly three classes ofparasites which can cause infections, at least in humans, protozoa(causing a protozoan infection), helminths (causing an helminthiasis)and ectoparasites. As it is known in the art, parasites have theintrinsic ability, upon infecting their host, to upregulate or enhanceTreg's levels and/or activity and thereby induce a state of immunetolerance. This is exemplified by filarial nematodes in which thenematode secretes substances that cause an increase in the host's Treglymphocytes levels. The increase in Tregs actively down-regulate theTh1, Th17 and Th2 responses necessary for eradication of the parasite.Administration of an agent that can reverse the parasite's induced Tregincrease would enhance the ability of the subject's immune system toeradicate the parasitic infection. In another embodiment, the infectionis caused by a virus (such as, for example, the human immunodeficiencyvirus or HIV) and, in such instance, it is referred to as a “viral”infection. In some embodiments, the viral infection is an acquiredimmunodeficiency syndrome or AIDS. In yet another embodiment, theinfection is caused by a bacterium (such as, for example, from aStreptococcus sp. (e.g., Streptococcus pneumoniae) and, in suchinstance, it is referred to as a “bacterial” infection. In someembodiments, the bacterial infection is pneumonia. As it is known in theart, Tregs are implicated in improving clearance and reducing injury dueto bacteria/viruses as well as increasing infections in viruses andbacteria. Viral and bacterial infections spread can be facilitated by anoverly strong immune response, hence Tregs would reduce this risk.However, elevated Treg, in the absence of a proinflammatory response,would cause a state of immune suppression. In another embodiment, theinfection is caused by a fungus and, in such instance, it is referred toas a “fungal” infection. Fungal infections are opportunistic and T cellsplay a critical role in stimulating the neutrophils which are able tolimit or clear the fungal infection. Subjects with a reduced (low orinappropriate) immune response have an increased risk towards fungalinfections (e.g., Aspergillus sp. (e.g. Aspergillus histoplasmosis) andCandidia sp. (e.g., Candida albicans)).

Leukocyte. As used herein, a leukocyte (also spelled leucocyte) isdefined as a blood cell lacking hemoglobin and having a nucleus.Leukocytes are produced and derived from hematopoietic stem cells.Leukocytes are also referred to as white blood cells. Leukocytes includegranulocytes (also known as polymorphonuclear leucocytes), e.g.,neutrophils, basophils and eosoniphils. Leukocytes also includeagranulocytes (or mononuclear leucocytes), e.g., lymphocytes, monocytesand macrophages. Some of the lymphocytes, referred to as T cells (orT-cell), bear on their surface a T-cell receptor. T cells are broadlydivided into cells expressing CD4 on their surface (also referred to asCD4-positive cells) and cells expressing CD8 on their surface (alsoreferred to as CD8-positive cells). Some of the lymphocytes, referred toas B cells (or B-cells), bear on their surface a B-cell receptor.

Low-immunogenic biocompatible polymer. As used herein, a“low-immunogenic polymer” refers to a polymer which is not or isunlikely to elicit an immune response in an individual. Thislow-immunogenic polymer is also capable, when grafted at the appropriatedensity, of masking antigenic determinants of a cell and lowering oreven preventing an immune response to the antigenic determinant when theantigenic determinant is introduced into a subject. A “biocompatiblepolymer” refers to a polymer which is non-toxic when introduced into asubject. Exemplary low-immunogenic biocompatible polymers includes, butare not limited to, polyethylene glycol (for examplemethoxypoly(ethylene glycol)), hyperbranched polyglycerol (HPG),2-alkyloxazoline (POZ) such as, for example, polyethyloxazoline (PEOZ)(Kyluik-Price D. L. et al. (2014)).

Non-proliferative leukocyte. As used herein, the term “non-proliferativeleukocyte” refers to a leukocyte which has been modified to no longerbeing capable of cellular proliferation (e.g. performing at least onecomplete division cycle). In some embodiments, this modification may betemporary and the non-proliferative properties of a leukocyte may belimited in time. For example, when a leukocyte is modified from acontact with a pharmacological agent capable of limiting itsproliferation, the removal of the pharmacological agent from the cellculture can allow the leukocyte to regain its proliferative properties.In other embodiments, the modification is permanent and the modifiedleukocyte cannot regain its proliferative properties. For example, whena leukocyte is irradiated, it is not possible for it to regain itsproliferative properties. In the context of the present application, theexpressions “non-proliferative leukocyte” or “leukocyte limited fromproliferating” are used interchangeably.

Peripheral blood mononuclear cells (PBMC). This term refers to the cellpopulation recuperated/derived from the peripheral blood of a subject(usually a mammal such as a human). PBMC usually contains T cells, Bcells and antigen presenting cells.

Pharmaceutically effective amount or therapeutically effective amount.These expressions refer to an amount (dose) of an acellular preparationeffective in mediating a therapeutic benefit to a patient (for exampleprevention, treatment and/or alleviation of symptoms of animmune-associated disorder or condition in which the ratio of Tregs topro-inflammatory T cells is high or low when compared to sex- andaged-matched healthy subjects). It is also to be understood herein thata “pharmaceutically effective amount” may be interpreted as an amountgiving a desired therapeutic effect, either taken in one dose or in anydosage or route, taken alone or in combination with other therapeuticagents.

Prevention, treatment and alleviation of symptoms. These expressionsrefer to the ability of the acellular preparation to limit thedevelopment, progression and/or symptomology of an immune-associateddisorder. In some embodiments, the immune-associated disorders areconditions caused/exacerbated by a low or inappropriate immune response(also known as a state of anergy or tolerance). The subjects beingafflicted with these conditions/disorders have a ratio of Tregs topro-inflammatory T cells which is considered high when compared to sex-and aged-matched healthy subjects. In such embodiment, the prevention,treatment and/or alleviation of symptoms encompasses decreasing thelevels of Treg cells and/or increasing the levels of pro-inflammatory Tcells. The acellular-based preparation is considered effective orsuccessful for treating and/or alleviating the symptoms associated withthe disorder when a reduction in the pro-tolerogenic state (whencompared to an untreated and afflicted individual) in the treatedindividual (previously known to be afflicted with the disorder) isobserved. A method or acellular-based preparation is consideredeffective or successful for preventing the disorder when a reduction inthe pro-tolerogenic state (when compared to an untreated and afflictedindividual) in the treated individual is observed upon an immunologicalchallenge (such as, for example, an antigenic challenge).

In another embodiment, the immune-associated disorders are conditionscause/exacerbated by an abnormal/excessive immune response (also known apathological inflammation). The subjects being afflicted with theseconditions/disorders have a ratio of Tregs to pro-inflammatory T cellswhich is considered low when compared to sex- and age-matched healthysubjects. In such embodiment, the prevention, treatment and/oralleviation of symptoms encompasses increasing the levels of Treg cellsand/or decreasing the levels of pro-inflammatory T cells. The acellularpreparation is considered effective or successful for treating and/oralleviating the symptoms associated with the disorder when a reductionin the pro-inflammatory state (when compared to an untreated andafflicted individual) in the treated individual (previously known to beafflicted with the disorder) is observed. The acellular-basedpreparation is considered effective or successful for preventing thedisorder when a reduction in the pro-inflammatory state (when comparedto an untreated and afflicted individual) in the treated individual isobserved. In instances where the conditions to be treated is cancer,exemplary symptoms which can be alleviated with the acellular-basedpreparations described herewith include, but are not limited to, numberand/or size of metastatic tumors, presence and/spread of metastatictumors and/or size of primary tumor. In instances where the conditionsto be treated is an infection, exemplary symptoms which can bealleviated with the acellular-based preparations described herewithinclude, but are not limited to, infectious agent's burden, infectiousagent's presence and fever.

Pro-inflammatory T cells. In the present context, pro-inflammatory Tcells are a population of T cells capable of mediating an inflammatoryreaction. Pro-inflammatory T cells generally include T helper 1 (Th1 orType 1) and T helper 17 (Th17) subsets of T cells. Th1 cells partnermainly with macrophage and can produce interferon-γ, tumor necrosisfactor-β, IL-2 and IL-10. Th1 cells promote the cellular immune responseby maximizing the killing efficacy of the macrophages and theproliferation of cytotoxic CD8+ T cells. Th1 cells can also promote theproduction of opsonizing antibodies. T helper 17 cells (Th17) are asubset of T helper cells capable of producing interleukin 17 (IL-17) andare thought to play a key role in autoimmune diseases and in microbialinfections. Th17 cells primarily produce two main members of the IL-17family, IL-17A and IL-17F, which are involved in the recruitment,activation and migration of neutrophils. Th17 cells also secrete IL-21and IL-22.

Proliferation-associated disorders. These disorders (also referred to ashyperproliferative disorders) form a class of diseases where cellsproliferate more rapidly, and usually not in an ordered fashion, thancorresponding healthy cells. The proliferation of cells causes anhyperproliferative state that may lead to biological dysfunctions, suchas the formation of tumors (malignant or benign). One of theproliferation-associated disorders is cancer. Also known medically as amalignant neoplasm, cancer is a term for a large group of differentdiseases, all involving unregulated cell growth. In cancer, cells divideand grow uncontrollably, forming malignant tumors, and invade nearbyparts of the body. The cancer may also spread to more distant parts ofthe body through the lymphatic system or bloodstream. In an embodiment,the cancer is a carcinoma (e.g. a cancer of the epithelial cells). Othertypes of cancer include, but are not limited to sarcoma, lymphoma,leukemia, germ cell tumor and blastoma.

Regulatory T cells. Regulatory T cells are also referred to as Treg andwere formerly known as suppressor T cell. Regulatory T cells are acomponent of the immune system and suppress immune responses of othercells. Regulatory T cells usually express CD3, CD4, CD8, CD25, andFoxp3. Additional regulatory T cell populations include Tr1, Th3,CD8⁺CD28⁻, CD69⁺, and Qa-1 restricted T cells. It has been recentlyshown that CD69 can exert regulatory function in the immune response bypreventing pro-inflammatory conditions. Under normal conditions, thisregulatory effect of CD69 is desired, but when expressed in the contextof a pro-inflammatory response to, for example, a tumor cell mass, willresult in impaired killing of the abnormal cells and diseaseprogression. Regulatory T cells actively suppress activation of theimmune system and prevent pathological self-reactivity, i.e. autoimmunedisease. The critical role regulatory T cells play within the immunesystem is evidenced by the severe autoimmune syndrome that results froma genetic deficiency in regulatory T cells. The immunosuppressivecytokines TGF-β and Interleukin 10 (IL-10) have also been implicated inregulatory T cell function. Similar to other T cells, a subset ofregulatory T cells can develop in the thymus and this subset is usuallyreferred to as natural Treg (or nTreg). Another type of regulatory Tcell (induced Treg or iTreg) can develop in the periphery from naïveCD4⁺ T cells. The large majority of Foxp3-expressing regulatory T cellsare found within the major histocompatibility complex (MHC) class IIrestricted CD4-expressing (CD4⁺) helper T cell population and expresshigh levels of the interleukin-2 receptor alpha chain (CD25). Inaddition to the Foxp3-expressing CD4⁺CD25⁺, there also appears to be aminor population of MHC class I restricted CD8⁺Foxp3-expressingregulatory T cells. Unlike conventional T cells, regulatory T cells donot produce IL-2 and are therefore anergic at baseline. Moreover,regulatory T cell produce elevated levels of IL-10 and TGF-β whichinhibit pro-inflammatory responses An alternative way of identifyingregulatory T cells is to determine the DNA methylation pattern of aportion of the foxp3 gene (TSDR, Treg-specific-demethylated region)which is found demethylated in Tregs.

Splenocytes. This term refers to the cell population obtained from thespleen of a subject (usually a mammal such as a rodent). Splenocytesusually comprise T cell, B cell as well as antigen presenting cells.

Syngeneic cell. A cell is considered “syngeneic” with respect to asubject (or a cell derived therefrom) if it is sufficiently identical tothe subject so as to prevent an immune rejection upon transplantation.Syngeneic cells are derived from the same animal species.

Viable. In the present context, the term “viable” refers to the abilityof a cell to complete at least one cell cycle and, ultimatelyproliferate. A viable cell is thus capable of proliferating. Byopposition, the term “non-viable” or “non-proliferative” both refer to acell which is no longer capable of completing at least one cell cycle.By comparison, the term “cycle arrest” refers to a cell which has beentreated to halt its cell cycle progression (usually with apharmacological agent) but which is still capable of re-entering thecell cycle (usually when the pharmacological agent is removed).

Xenogeneic cell. A cell is considered “xenogeneic” with respect to asubject (or a cell derived from the subject) when it is derived from adifferent animal species than the subject. A xenogeneic cell is expectedto be rejected when transplanted in an immunocompetent host.

BRIEF DESCRIPTION OF THE DRAWINGS

Having thus generally described the nature of the invention, referencewill now be made to the accompanying drawings, showing by way ofillustration, a preferred embodiment thereof.

FIGS. 1A to C illustrate the effects of size (MW) separation and RNasetreatment on the immunomodulary effects of acellular preparations.Unmodified conditioned murine plasma (obtained from donor mice 5 dayspost splenocyte transfer), size fractionated-conditioned murine plasmaor RNase-treated conditioned murine plasma was administered once tonaïve mice and Treg/Th17 levels were measured (when) in the spleen. (A)Results are shown as the percentage of Th17 cells (in function of CD4+cells) in function of type of conditioned medium (white bars=conditionedplasma obtained from administering saline, hatched bars=conditionedplasma obtained from administering unmodified allogeneic splenocytes,grey bars=conditioned plasma obtained from administeringpolymer-modified allogeneic splenocytes) and size fractionation(non-fractioned or complete conditioned serum, fraction>100 kDa,fraction between 30 and 100 kDa, fraction between 10 and 30 kDa,fraction<10 kDa). a denotes the mean value for unfractionatedconditioned medium prepared from mice previously treated with unmodifiedallogeneic cells. b denotes the mean value for unfractionatedconditioned medium prepared from mice previously treated withmPEG-modified allogeneic cells. (B) Results are shown as the percentageof Treg cells (in function of CD4+ cells) in function of type ofconditioned medium (white bars=conditioned plasma obtained fromadministering saline, hatched bars=conditioned plasma obtained fromadministering unmodified allogeneic splenocytes, grey bars=conditionedplasma obtained from administering polymer-modified allogeneicsplenocytes) and size fractionation (non-fractioned or completeconditioned serum, fraction>100 kDa, fraction between 30 and 100 kDa,fraction between 10 and 30 kDa, fraction<10 kDa). a denotes the meanvalue for unfractionated conditioned medium prepared from micepreviously treated with unmodified allogeneic cells. b denotes the meanvalue for unfractionated conditioned medium prepared from micepreviously treated with mPEG-modified allogeneic cells. (C) Results areshown as the percentage of Treg cells (in function of CD4⁺ cells, leftpanel) or Th17 cells (in function of CD4⁺ cells, right panel) infunction of type of treatment (white bars=N=naïve untreated animals;grey bars=AC=unmodified allogeneic cells; diagonal hatchbars=conditioned plasma obtained from administered unmodifiedsplenocytes treated (allo-plasma (+)) or not (allo-plasma (−)) withRNase; horizontal hatch bars=conditioned plasma obtained fromadministering polymer modified splenocytes treated (mPEG-allo-plasma(+)) or not (mPEG-allo-plasma (−)) with RNase.

FIGS. 2A to C illustrate the cellular modulation in Treg cells upon theadministration of condition murine plasma. Saline, unmodifiedconditioned plasma (obtained by administering saline to the animal,identified as plasma (saline)), conditioned plasma obtained from theadministration of non-modified allogeneic cells (identified as plasma(allo)) or the conditioned plasma obtained from the administration ofpolymer-modified allogeneic cells (identified as plasma (mPEG-Allo)) wasinjected once (1) or thrice (3) in the animals. After 5 days, theanimals were sacrificed and their spleen and brachial lymph nodes wereobtained. (A) Results are shown as the percentage of CD4⁺CD25⁺ T cellsin function of type of conditioned plasma administered in the spleen(white bars) and in the brachial lymph nodes (grey bars). * denotesp<0.001 relative to treatment with conditioned plasma from mice treatedwith saline, # denotes p<0.001 relative to treatment with conditionedmedium derived from mice treated with unmodified allogeneic splenocytes.(B) Results are shown as the percentage of CD69⁺CD4⁺CD25⁻ T cells infunction of type of conditioned plasma administered in the spleen (whitebars) and in the brachial lymph nodes (grey bars). * denotes p<0.001relative to treatment with conditioned plasma from mice treated withsaline, # denotes p<0.001 relative to treatment with conditioned mediumderived from mice treated with unmodified allogeneic splenocytes. (C)Results are shown as the percentage of Foxp3⁺, CD25⁺ or CD69⁺ of CD4⁺cells in function of the conditioned plasma administered in spleniccells (white bars) and lymph node cells (gray bars).

FIGS. 3A to E illustrate the size fractionated conditioned plasma on theintracellular expression of cytokines. Unmodified conditioned murineplasma (obtained from donor mice 5 days post saline or splenocytetransfer), size fractionated-conditioned murine plasma was administeredonce to naïve mice and Treg/Th17 levels were measured (when) in thespleen. Results are shown as the percentage intracellular cytokinepositive CD4⁺ cells in function of type of conditioned medium (whitebars=conditioned plasma obtained from administering saline, hatchedbars=conditioned plasma obtained from administering unmodifiedallogeneic splenocytes, grey bars=conditioned plasma obtained fromadministering polymer modified allogeneic splenocytes) and sizefractionation (non-fractioned or complete conditioned serum,fraction>100 kDa, fraction between 30 and 100 kDa, fraction between 10and 30 kDa, fraction<10 kDa) for (A) IL-10, (B) IL-2, (C) IFN-γ, (D)TNF-α and (E) IL-4. * denotes p<0.001 relative to treatment withconditioned plasma from mice treated with saline, # denotes p<0.001relative to treatment with conditioned medium derived from mice treatedwith unmodified allogeneic splenocytes.

FIGS. 4A to E illustrates the in vivo effects of the various conditionedmedium and preparations derived therefrom on the intracellularexpression of cytokines as well as type of CD4⁺ cells. Conditionedplasma was obtained by administering naïve mice with saline, unmodifiedallogeneic splenocytes or polymer-modified allogeneic splenocytes (PEG)and recuperating plasma after 5 days. The obtained plasma was eitheradministered directly (•=untreated) or optionally treated with RNaseA(∘=conditioned plasma, ▪=miRNA enriched fraction of conditioned plasma)and/or further purified so as to retain and enrich the <10 kDa fraction(e.g. miRNA) (▪=untreated miRNA, □=RNase A-treated miRNA) prior toadministration. As a control, RNase A was also administered directly tosome animals. After 30, 60, 120, 180, 270 days, animals were sacrificed,their spleen was removed and CD4⁺ cells were characterized by flowcytometry. Results are shown for intracellular cytokine expression: IL-2(A), INF-γ (B), IL-10 (C), as well as CD4⁺ cell type: Treg (Foxp3⁺) (D)and Th17 (IL-17+) (E) CD4⁺ cells.

FIGS. 5A to D illustrates the effects of the TA and IA preparations onthe phosphorylation of phosphokinases of resting Jurkat cells. Resultsare shown as fold modulation (when compared to saline-treated Jurkatcells) for each kinase tested. Results for TA preparations are shown inpanels (A) to (C). Comparative results between TA and IA preparationsare shown in panels (D). (A) On this panel, Akt is considered to besignificantly increasingly phosphorylated in the presence of the TA1preparation. (B) On this panel, PRAS40 is considered to be significantlyincreasingly phosphorylated, in the presence of the TA1 preparation. (C)On this panel, HSP60 is considered to be significantly decreasinglyphosphorylated, in the presence of the TA1 preparation. (D) On thispanel, it can be seen that the phosphorylation of kinases HSP60, WNK1,STAT3, RSK1/2/3, p53 and Akt are inversely modulated in the presence ofTA1 preparations (white bars) and IA preparations (grey bars). * denotesgreater than 10-fold increase in protein phosphorylation over restingJurkat cells. # denotes greater than 10-fold decrease in proteinphosphorylation over resting Jurkat cells.

FIG. 6 illustrates the in vitro effects of the murine IA1 preparationson human PBMCs. Murine TA1 or IA1 preparations (either 25 μL, 50 μL, 100μL or 200 μL) were included in a human PBMC MLR assay and cellularproliferation was measured. Results are shown as percent inproliferation (CD3⁺CD4⁺ cells) in function of conditions (Rest=restingMLR, MLR=conventional MLR without TA1, Murine TA-1=MLR with TA1, Murine1A-1=MLR with IA1) and TA1/IA1 concentration (in μL) after 10 days (A)or 14 days (B). # denotes p<0.001 relative to conventional MLR value andand * denotes p<0.001 relative to murine IA1 MLR. ¢ denotes theconcentration of the TA1 or IA1 preparation used in the in vivo mousestudy (e.g., FIG. 7).

FIGS. 7A to D illustrates the in vivo effects of the sequential combineduse of murine TA1 and IA1 preparations on the level of Treg and Th17cells. Naïve animals were divided in two groups: those receiving asingle type of preparation (Saline, AI1 or TA1 preparations) and thosereceiving two types of preparations (1° TAI/2° IA1 or 1° IA1/2° TA1).All animals were administered thrice (at day 0, 2 and 4) with saline,the TA1 preparation or the IA1 preparation. Animals receiving a secondpreparation were administered thrice (at day 9, 11 and 13) with the IA1or the TA1 preparation. At day 40, all animals were sacrificed and thelymphocytes in their spleen and brachial lymph node were characterized.In (A) and (B), results are shown as the percentage of Treg cells (withrespect to the total number of CD4⁺ cells) in function of treatment inthe spleen (A) and the brachial lymph nodes (B). Δd refers to theincrease in Treg cell levels between naïve animals and those havingreceived TA1 preparations. Δd′ refers to the decrease in Treg celllevels between naïve animals and those having received IA1 preparations.Δd′^(2o) refers to the decrease in Treg cell levels between animalshaving received only TA1 preparations and those having received TA1preparations followed by IA1 preparations. Δd^(2o) refers to theincrease in Treg cell levels between animals having received only IA1preparations and those having received IA1 preparations followed by TA1preparations. The gray zone in this figure indicates naïve Treg levels.The dashed lines indicate the maximal Treg levels (obtained with TA1preparations) and minimal Treg level (obtained with IA1 preparations).In (C) and (D), results are shown as the percentage of Th17 cells (withrespect to the total number of CD4⁺ cells) in function of treatment inthe spleen (C) and the brachial lymph nodes (D). Δd refers to thedecrease in Th17 cell levels between naïve animals and those havingreceived TA1 preparations. Δd′ refers to the increase in Th17 celllevels between naïve animals and those having received IA1 preparations.Δd′^(2o) refers to the increase in Th17 cell levels between animalshaving received only TA1 preparations and those having received TA1preparations followed by IA1 preparations. Δd^(2o) the decrease in Th17cell levels between animals having received only IA1 preparations andthose having received IA1 preparations followed by TA1 preparations. Thegray zone in this figure indicates naïve Th17 levels. The dashed linesindicate the minimal Th17 levels (obtained with TA1 preparations) andmaximal Th17 level (obtained with IA1 preparations).

FIGS. 8A to 8C provide a comparison of the miRNA populations betweendifferent MLR assays. A human PBMC MLR assay (using unmodified (controlMLR) or polymer modified leukocyte (mPEG MLR)) was conducted and miRNAcontent was partially determined. Volcano plots of comparing the miRNApopulation of the conditioned medium of the control MLR to the one ofthe supernatant of resting cells (A), comparing the miRNA population ofthe conditioned medium of a mPEG MLR to the one of the supernatant ofresting cells (B) and comparing the miRNA population of the conditionedmedium of a mPEG MLR to the one of the conditioned medium of a controlMLR (C) are provided. Results are provided in −Log₁₀ (p value) infunction of Log₂ fold change. In these volcano plots, the followingmiRNAs have been identified with numbers:

-   -   1 has-miR-298    -   2 has-miR-34a-5p    -   3 has-miR-574-3p    -   4 has-miR-125b-5p    -   5 has-let-7a-5p    -   6 has-miR-196a-5p    -   7 has-miR-148a-3p    -   8 has-let-7e-5p    -   9 has-miR-134

FIG. 9 provides a partial miRNA compositional analysis of theconditioned medium of a mPEG MLR (white bars) and of a control MLR(black bars). Results are provided, for each miRNA, as log₂ foldregulation when compared to the miRNA present in the supernatant ofresting cells. White open stars denote Log₂-fold change and black solidstars denote significant changes in volcano plot analysis.

FIG. 10 provides a selection of the miRNA compositional analysis of theconditioned medium of a mPEG MLR (white bars) and of a control MLR(black bars). Results are provided, for each miRNA, as log₂ foldregulation when compared to the miRNA present in the supernatant ofresting cells. White open stars denote Log₂-fold change and black solidstars denote significant changes and or clustergram (heatmap) determinedmiRNA shifts denoted in volcano plot analysis.

DETAILED DESCRIPTION

In accordance with the present disclosure, there is provided atherapeutic combination for modulating the level of regulatory T cellsand/or the level of pro-inflammatory T cells for ultimatelyintentionally inducing immune modulation in a subject in need thereof.The acellular-based pro-inflammatory preparations are obtained bycontacting at least two distinct leukocyte populations which areconsidered allogeneic with respect to one another. The therapeuticcombinations described herein comprise at least one acellularpro-inflammatory preparation and at least one acellular pro-tolerogenicpreparation. The pro-inflammatory preparation can be obtained bycontacting the two allogeneic leukocyte populations under conditions toallow pro-inflammatory allo-recognition but to limit or preventpro-tolerogenic recognition. The pro-tolerogenic preparations can beobtained by contacting the two allogeneic leukocyte populations underconditions to allow pro-tolerogenic allo-recognition but to limit orprevent pro-inflammatory recognition. In the process for making thepro-tolerogenic preparation, one of the two leukocyte population hasbeen modified with a polymer. For either acellular preparations, thecontact between the two types of leukocytes can occur in vitro, ex vivoor in vivo. The biological fluid (cell culture medium or fractionthereof, blood, blood fraction) in which the two types of leukocyteshave been contacted is then recuperated in RNase-free conditions and canbe used, without further purification to induce an immune modulation.

Since the acellular preparations can optionally be enriched in miRNAs,it is important that the cell culture and/or the blood/blood fraction beprocessed in conditions so as to retain the integrity of the majority ofthe miRNA species present, for example by substantially inhibiting RNAdegradation. As used herein, the term “substantially inhibiting RNAdegradation” indicate that the conditions allow for the degradation ofless than 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%,7%, 6% or 5% of the miRNA population obtained by RNases. RNases include,but are not limited endoribonucleases (e.g., RNase A, RNase H, RNase I,RNase III, RNase L, RNase P, RNase PhyM, RNase T1, RNase T2, RNase U2,RNase V1 and/or RNase V) and exoribonucleases (e.g., polynucleotidepPhosphorylase (PNPase), RNase PH, RNase II, RNase R, RNase D, RNase T,Oligoribonuclease, Exoribonuclease I and/or Exoribonuclease II). Sinceit is known in the art that miRNAs are, in general, more resistancetowards degradation than messenger RNAs, the conditions for obtainingand processing the cell culture/blood can allow for some RNAdegradation, preferably limited to the mRNA fraction.

As it will be shown below, acellular preparations obtained fromallogeneic leukocytic cells provides a significant opportunity tomodulate the responsiveness (i.e., immunoquiescent versuspro-inflammatory) of the recipient's immune system. The therapeuticcombinations described herein can be used to intentionally induce apro-tolerogenic state in an afflicted subject and, afterwards, provideimmune stimulation to the same subject. Optionally, the therapeuticcombination can also be used to intentionally re-induce apro-tolerogenic state in the same subject. Alternatively, thetherapeutic combinations described herein can be used to intentionallyinduce a pro-inflammatory state in an afflicted subject and, afterwards,provide immune tolerance to the same subject. Optionally, thetherapeutic combination can also be used to re-induce a pro-inflammatorystate in the same subject.

Therapeutic Combinations and Associated Therapeutic Kits

The therapeutic combinations described herein comprise at least onepro-tolerogenic acellular preparation and at least one pro-inflammatoryacellular preparation. The pro-tolerogenic acellular preparation and thepro-inflammatory acellular preparation are not to be administered in asimultaneous manner, but in a sequential manner. A first preparation canbe administered to the afflicted subject (e.g., which, in an embodiment,is naïve to the acellular pro-tolerogenic and/or pro-inflammatorypreparations described herein) to modulate his initialTregs/pro-inflammatory T cells ratio (e.g., which has been determined tobe associated with an immune disorder) to a first Tregs/pro-inflammatoryT cells ratio (e.g., believed to be beneficial for preventing, treatingor alleviations the symptoms associated with an immune disorder). Thesecond preparation can then administered afterwards either to achieve asecond Tregs/pro-inflammatory T cell ratio (e.g., usually between theinitial ratio and the first ratio) or to revert back to the initialTregs/pro-inflammatory T cells ratio (of the naïve subject). Optionally,the therapeutic combinations described herein can also comprise a thirdpreparation for achieving a third Treg/pro-inflammatory T cell ratio.

In some embodiments, the acellular pro-tolerogenic preparation isadapted to initially be administered to the subject in need thereofprior to the administration of the acellular pro-inflammatorypreparation. In such instance, a first state of immune tolerance isinduced in the treated subject (by the administration of a first dose ormultiple doses of the acellular pro-tolerogenic preparation) and then,when it is determined that a further increase in the immune response iswarranted, an immune stimulation is then induced in the treated subject(by the administration of a first dose or multiple doses of theacellular pro-inflammatory preparation). It is possible that, in somesituations, it is warranted to return to an increased state of tolerancein the treated subject after the onset of an immune stimulation. In suchinstance, a further state of immune tolerance can be induced by theadministration of a second dose (or multiple doses) of the acellularpro-tolerogenic preparation to the treated subject. In still furtherembodiments, it is possible to induce a further state of immunestimulation to the treated subject by administering a second dose (or asecond round of doses) of the acellular pro-inflammatory preparation. Assuch, it is possible to provide a therapeutic combination comprising atleast two, at least three, at least four, at least five or more doses ofthe acellular pro-tolerogenic preparations and at least one, at leasttwo, at least three, at least four, at least five or more of theacellular pro-inflammatory preparation that are being to be administeredin a sequential manner. In one embodiment, the acellular pro-tolerogenicpreparations are administered in an alternate fashion with the acellularpro-inflammatory preparations. In another embodiment, more than one doseof the acellular pro-tolerogenic preparation are first administeredsequentially and then, at least one dose (or more than one dose) of thepro-inflammatory preparation(s) is(are) administered.

In other embodiments, the acellular pro-inflammatory preparation isadapted to be initially administered to the subject in need thereofprior to the administration of the acellular pro-tolerogenicpreparation. In such instance, a first state of immune stimulation isinduced in the treated subject (by the administration of a first dose ormultiple doses of the acellular pro-inflammatory preparation) and then,when it is determined that a decrease in the immune response iswarranted, an immune tolerance state is then induced in the treatedsubject (by the administration of one or more doses the acellularpro-tolerogenic preparation). It is possible that, in some situations,it is warranted to return to an increased state of stimulation in thetreated subject after the onset of an immune tolerance. In suchinstance, a further state of immune stimulation is induced by theadministration of a second dose (or multiple doses) of the acellularpro-inflammatory preparation to the treated subject. In still furtherembodiments, it is possible to induce a further state of immunetolerance to the treated subject by administering a second dose of theacellular pro-tolerogenic preparation. As such, it is possible toprovide a therapeutic combination comprising at least two, at leastthree, at least four, at least five or more doses of the acellularpro-inflammatory preparations and at least one, at least two, at leastthree, at least four, at least five or more of the acellularpro-tolerogenic preparation that are being to be administered in asequential manner. In one embodiment, the acellular pro-inflammatorypreparations are administered in an alternate fashion with the acellularpro-tolerogenic preparations. In another embodiment, more than one doseof the acellular pro-inflammatory preparation are first administeredsequentially and then, at least one dose (or more than one dose) of thepro-tolerogenic preparation(s) is(are) administered.

The present disclosure also provides therapeutic kits comprising thetherapeutic combinations described herein. The therapeutic kit comprisesat least one, at least two, at least three, at least four, at least fiveor more doses of the acellular pro-tolerogenic described herein, atleast one, at least two, at least three, at least four, at least five ormore doses of the acellular pro-inflammatory preparation describedherein as well as instructions for using the acellular pro-tolerogenicpreparations and the acellular pro-inflammatory preparations in asequential and, optionally alternate, manner. The instructions canspecify, for example, that the acellular pro-tolerogenic preparation isinitially to be administered to the subject prior to the administrationof the acellular pro-inflammatory preparation. Alternatively, theinstructions can specify that the acellular pro-inflammatory preparationis initially to be administered to the subject prior to the acellularpro-tolerogenic preparation. The therapeutic kits can also providedistinct containers for each acellular preparation to avoid the physicalcontact between each type of preparations (e.g. pro-tolerogenic vs.pro-inflammatory) or each dose of the same type of preparations. Thetherapeutic kits can also provide means for administering thepreparations to the subject, such as, for example, means for deliveringintravenously the preparations (such as syringes). In some embodiments,the therapeutic kits can provide a syringe for each dose of acellularpreparation that needs to be administered.

The preparations of the therapeutic kits can be formulated in afreeze-dried form destined to be reconstituted with a pharmaceuticallyacceptable excipient (such as a physiological saline solution).Alternatively, the preparations of the therapeutic kits can beformulated in a solution which would stabilize or limit miRNAdegradation (e.g., ethanol for example) destined to be diluted with apharmaceutically acceptable excipient (e.g., saline for example). Thetherapeutic kits can also comprise the pharmaceutically acceptableexcipient for reconstituting the freeze-dried preparations or fordiluting the solution containing the preparations, optionally dividedinto pre-measured volumes for reconstituting/diluting a singlepreparation.

The therapeutic kits can also comprise other components which wouldallow to determine the ratio in the level of regulatory T (Treg) cellsto the level of pro-inflammatory T cells in the subject intended to betreated. For example, the therapeutic kits can comprise a first set oflabeled (e.g., fluorescent tagged) antibodies to detect the number ofTreg cells (eg., anti-CD25, anti-CD69 and/or anti-FoxP3⁺ antibodies)cells and a second set of labeled (e.g., fluorescent tagged) antibodiesto detect pro-inflammatory T cells (e.g., Th17 or Th1). The therapeutickits could also comprise anti-CD4 antibodies to determine the number ofCD4⁺ T cells (Treg and pro-inflammatory T cells) in the sample obtainedfrom the subject intended to be treated. In yet another embodiment, thekit could contain an algorithm card to determine the immune state of thesubject (e.g., pro-inflammatory vs. pro-tolerogenic) or how much of thepro-inflammatory or pro-tolerogenic preparations need to be administeredto the subject in order to achieve the desired therapeutic target.

(i) Process for Obtaining the Acellular Pro-Tolerogenic Preparation

The acellular pro-tolerogenic preparations presented described hereincan be obtained by contacting two distinct and allogeneic leukocytepopulations (referred herein to the first leukocyte and the secondleukocyte). The two leukocyte populations are contacted under conditionsso as to allow (and in some embodiments to favor) pro-tolerogenicallo-recognition and to prevent (and in some embodiments to inhibit)pro-inflammatory allo-recognition.

Prior to the contact between the leukocyte populations, at least one ofthe first and/or the second leukocyte is modified to bear on theirsurface a low-immunogenic polymer. It is important that the polymeradded or the conditions used to graft the polymer do not significantlyalter the ability of the two leukocyte populations to mediate apro-tolerogenic allo-recognition. It is important that the polymer usedexhibits both low-immunogenicity and biocompatibility once introducedinto a cell culture system or administered to the test subject.Polyethylene glycol (particularly methoxypoly(ethylene glycol)),2-alkyloxazoline (POZ) such as, for example and polyethyloxazoline(PEOZ) and hyperbranched polyglycerol (HPG) are exemplary polymers whichall exhibit low immunogenicity and biocompatibility and can besuccessfully used to modify the first leukocyte (and optionally thesecond leukocyte). In some embodiments, it is preferable to use a singletype of polymer to modify the surface of leukocytes. In otherembodiments, it is possible to use at least two distinct types ofpolymers to modify the surface of the leukocyte.

In an embodiment, the low-immunogenic biocompatible polymer can becovalently associated with the membrane-associated protein(s) of theleukocyte by creating a reactive site on the polymer (for example bydeprotecting a chemical group) and contacting the polymer with theleukocyte. For example, for covalently binding a methoxypoly(ethyleneglycol) to the surface of a leukocyte, it is possible to incubate amethoxypoly(-ethylene glycol) succinimidyl valerate (reactive polymer)in the presence of the leukocyte. The contact between the reactivepolymer and the leukocyte is performed under conditions sufficient forproviding a grafting density which will allow pro-tolerogenicallo-recognition and prevent pro-inflammatory allo-recognition. In anembodiment, the polymer is grafted to a viable leukocyte and underconditions which will retain the viability of the leukocyte. A linker,positioned between the surface of the leukocyte and the polymer, canoptionally be used. Examples of such polymers and linkers are describedin U.S. Pat. Nos. 5,908,624; 8,007,784 and 8,067,151. In anotherembodiment, the low-immunogenic biocompatible polymer can be integratedwithin the lipid bilayer of the cytoplasmic membrane of the leukocyte byusing a lipid-modified polymer.

As indicated above, it is important that the low-immunogenicbiocompatible polymer be grafted at a density sufficient allowingpro-tolerogenic allo-recognition while preventing pro-inflammatoryallo-recognition of the first leukocyte by the second leukocyte (andvice versa). In an embodiment, the polymer is polyethylene glycol (e.g.,linear) and has an average molecular weight between 2 and 40 KDa as wellas any combinations of molecular weight within this range. In anotherembodiment, the polymer is polyethylene glycol (e.g. linear) and has anaverage molecular weight between 2 and 40 KDa as well as anycombinations of molecular weight within this range. In a furtherembodiment, the average molecular weight of the PEG to be grafted is atleast 2, 3, 4, 5, 10, 15, 20, 25, 30, 35 or 40 kDa. In anotherembodiment, the average molecular weight of the PEG to be granted is nomore than 40, 35, 30, 25, 20, 15, 10, 5, 4, 3, or 2 kDa. In anotherembodiment, the grafting concentration of the polymer (per 20×10⁶ cells)is no more than 5.0, 4.5, 4.0, 3.5, 3.0, 2.5, 2.4, 2.0, 1.0, 0.5, 0.4,0.3, 0.2, 0.1, 0.05, 0.01 or 0.005 mM. In still another embodiment, thegrafting concentration of the polymer (per 20×10⁶ cells) is equal to orlower than 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 2.0, 2.4,2.5, 3.0, 3.5, 4.0, 4.5 or 5.0 mM. In embodiments where the polymer isgrafter to affect the viability of the leukocyte (for example bycreating cellular instability, cellular fragmentation or vesiculization,the concentration of the polymer (per 20×10⁶ cells) is equal to orhigher than 10 mM. In order to determine if pro-inflammatoryallo-recognition occurs, various techniques are known to those skilledin the art and include, but are not limited to, a standard mixedlymphocyte reaction (MLR), high molecular weight mitogen stimulation(e.g. PHA stimulation) as well as flow cytometry (Chen and Scott, 2006).In order to determine if a pro-tolerogenic allo-recognition occurs,various techniques are known to those skilled in the art and include,but are not limited to, the assessment of the level of expansion anddifferentiation of Treg cells and or prevention of Th17expansion/differentiation.

Before or after being modified with a low-immunogenic and biocompatiblepolymer, the first leukocyte can optionally be modified to refrain frombeing proliferative. This modification preferably occurs prior to itsintroduction in a cell culture system or its administration into a testsubject. For example, the leukocyte can be irradiated (e.g.γ-irradiation) prior to its introduction in a cell culture system or inthe test subject. Upon irradiation, the leukocyte is not consideredviable (e.g. capable of proliferation). In an embodiment, polymergrafting can affect the leukocyte viability and be used to refrain theleukocyte from proliferating. Alternatively, leukocyte can be treatedwith a pharmacological agent capable of halting cell cycle progression.Upon the administration of such pharmacological agent, the leukocyte isconsidered viable since it can resume cellular proliferation when theagent is removed from the cell-containing medium.

It is also contemplated that the second leukocyte (which can optionallybe modified with the low-immunogenic and biocompatible polymer) be alsooptionally modified to refrain from being proliferative. For example,the leukocyte can be irradiated (e.g. γ-irradiation) prior to itsintroduction in a cell culture system or in the test subject. Uponirradiation, the leukocyte is not considered viable (e.g. capable ofproliferation). In an embodiment, polymer grafting can affect theleukocyte viability and can be used to refrain the leukocyte fromproliferating. Alternatively, leukocyte can be treated with apharmacological agent capable of halting cell cycle progression. Uponthe administration of such pharmacological agent, the leukocyte isconsidered viable since it can resume cellular proliferation when theagent is removed from the cell-containing medium. However, when thesecond leukocyte is modified from being proliferative, it is importantthe first leukocyte with which it is being contacted remainsproliferative.

In order to generate the acellular pro-tolerogenic preparations, it isnot necessary to provide homogeneous leukocyte populations. For example,the first leukocyte population (such as, for example a PBMCs orsplenocytes) can be introduced in a cell culture system and contactedwith a second leukocyte population (such as, for example a PBMCs orsplenocytes) or administered to the test subject. However, in someembodiments, it is possible to provide and contact more homogeneousleukocyte populations. For example, the first leukocyte population canbe relatively homogenous (such as, for example, a T cell population) andintroduced in a cell culture system comprising a second leukocytepopulation (such as, for example a PBMC or splenocyte) or administeredto the test subject. In another example, the first leukocyte population(such as, for example a PBMC or splenocyte) can be introduced in a cellculture system comprising a second leukocyte population which can berelatively homogeneous (such as, for example, a T cell population). In afurther example, the first leukocyte population can be relativelyhomogenous (such as, for example, a T cell population) and introduced ina cell culture system comprising a second leukocyte population which canbe relatively homogeneous (such as, for example, a T cell population).

To provide the acellular pro-tolerogenic preparations describedherewith, the leukocytes used can be mature leukocytes or be provided inthe form of stem cells (e.g., for example non-embryonic stem cells). Forexample, leukocytes can be obtained from isolating peripheral bloodmononuclear cells (PBMC) from the subject. Optionally, the PBMCs can bedifferentiated in vitro into dendritic (DC) or DC-like cells.Alternatively, the leukocytes can be obtained from the spleen (e.g.,splenocytes). Leukocytes usually include T cells, B cells and antigenpresenting cells. In some embodiments, cells of sufficient antigenicvariation and immunogenicity are used. In addition, for providing theacellular pro-tolerogenic preparations, the leukocytes but noterythrocytes are necessary since the polymer-modified erythrocytes arenot capable of eliciting a pro-tolerogenic allo-recognition whenadministered in a test subject. However, traces of erythrocytes in theleukocyte population used are tolerated (for example, less than about10%, less than about 5% or less than about 1% of the total number ofcells in the preparation).

Even though it is not necessary to further purify the leukocytes toprovide the acellular pro-tolerogenic preparations, it is possible touse a pure cell population or a relatively homogenous population ofcells as leukocytes. This “pure” cell population and “relativehomogenous population” of cells can, for example, essentially consistessentially of a single cell type of T cells, B cells, antigenpresenting cells (APC) or stem cells. Alternatively, the population ofcells can consist essentially of more than one cell type. The populationof cells can be obtained through conventional methods (for example cellsorting or magnetic beads). In an embodiment, when the population ofcells consist of a single cell type (for example, T cells), thepercentage of the cell type with respect to the total population ofcells is at least 90%, at least 95% or at least 99%. The relativelyhomogenous population of cells is expected to contain some contaminatingcells, for example less than 10%, less than 5% or less than 1% of thetotal population of cells.

The first leukocyte and/or second leukocyte can be obtained from anyanimals, but are preferably derived from mammals (such as, for example,humans and mice). In an embodiment, the first or second leukocyte can beobtained from a subject intended to be treated with the acellularpro-tolerogenic preparation.

The first and/or second leukocyte can be expanded in vitro prior to theintroduction in a cell culture system or the administration to a testsubject.

As indicated above, the first and second leukocytes are contacted underconditions to allow pro-tolerogenic allo-recognition (e.g. expansion ofTreg cells and/or differentiation of naïve T cells in Treg cells) andprevent/inhibit pro-inflammatory allo-recognition (e.g. expansion ofpro-inflammatory T cells and/or differentiation of naïve T cells inpro-inflammatory T cells). When the contact occurs in vitro, it isimportant that the first leukocyte and the second leukocyte be culturedunder conditions allowing physical contact between the two leukocytepopulations and for a time sufficient to provide a conditionedpro-tolerogenic medium. As used herein, a conditioned pro-tolerogenicmedium refers to physical components of a cell culture (or fractionthereof, such as the cell culture supernatant) obtained by contactingthe first and the second leukocyte and having the pro-tolerogenicproperties described herein. Usually, the conditioned medium is obtainedat least 24 hours after the initial contact between the first and secondleukocyte. In some embodiments, the conditioned pro-tolerogenic mediumis obtained at least 48 hours or at least 72 hours after the initialcontact between the first and the second leukocyte. In an embodiment,the conditioned pro-tolerogenic medium can be obtained after at least 24hours of incubating the first leukocyte with the second leukocyte. Whenthe incubation takes place in a 24-well plate, the concentration of eachleukocyte population can be, for example, at least 1×10⁶ cells.

When the contact occurs in vivo, it is important that the firstleukocyte be administered to an immune competent test subject (bearingthe second leukocyte) and that the blood or blood fraction be obtainedat a later a time sufficient to provide a conditioned pro-tolerogenicblood. The test subject is a subject being immune competent and having aTreg/pro-inflammatory ratio which is substantially similar to age- andsex-matched healthy subjects. As used herein, the conditionedpro-tolerogenic blood refers to physical components present in the blood(or fraction thereof, such as the plasma) obtained by administering thefirst leukocyte to the immune competent test subject and having thepro-tolerogenic properties described herein. It is recognized by thoseskilled in the art that the conditioned pro-tolerogenic blood may beobtained more rapidly by increasing the amount of leukocytes beingadministered or administering more than once (for example one, twice orthrice) the first leukocyte. Usually, the conditioned pro-tolerogenicblood can be obtained at least one day after the administration of thefirst leukocyte. In some embodiment, the conditioned pro-tolerogenicblood is obtained at least 2, 3, 4, 5, 6 or 7 days after theadministration of the first leukocyte. In an embodiment, the conditionedpro-tolerogenic blood can be obtained by administering at least 5×10⁶allogeneic polymer-modified leukocytes to the test subject (e.g. testmouse) and recuperating the plasma five days later. In some embodiments,the conditioned pro-tolerogenic blood can be obtained by administeringat least 20×10⁶ allogeneic polymer-modified leukocytes to the testsubject (e.g. test mouse).

As indicated herein, the two leukocyte populations are consideredallogeneic (and in some embodiments, xenogeneic). When the acellularpro-tolerogenic preparation is obtained in vivo by, for example, aconditioned pro-tolerogenic blood/blood fraction can be obtained byadministering the first leukocyte to the test subject, the firstleukocyte can be allogeneic or xenogeneic to the test subject. In suchembodiment, it is also contemplated that the first leukocyte beautologous, syngeneic, allogeneic or xenogeneic to a treated subject whois going to receive the acellular pro-tolerogenic preparation. When theacellular pro-tolerogenic preparation is obtained in vitro by, forexample, a conditioned pro-tolerogenic medium can be obtained byco-culturing the first leukocyte with the second leukocyte, the firstleukocyte can be allogeneic or xenogeneic to the second leukocyte. Insuch embodiment, it is also contemplated that the first leukocyte beautologous, syngeneic, allogeneic or xenogeneic to a treated subject whois going to receive the acellular pro-tolerogenic preparation. Inaddition, it is also contemplated that the second leukocyte beautologous, syngeneic, allogeneic or xenogeneic to a treated subject whois going to receive the acellular pro-tolerogenic preparation.

Once the conditioned pro-tolerogenic preparation (medium or blood forexample), it can be further processed to substantially remove the cellsand cellular debris that can be present. This processing step can beachieved by submitting the conditioned pro-tolerogenic medium or theconditioned pro-tolerogenic blood to a centrifugation step and/or afiltration step. For example, blood can be processed as to obtain theplasma. Since the majority of the immuno-modulatory effects of theacellular pro-tolerogenic preparations reside in a fraction sensitive toribonucleic acid degradation (e.g., RNase degradation), this processstep should be conducted in conditions which would substantially limitor even inhibit ribonucleic acid degradation.

The conditioned pro-tolerogenic preparation can also be processed(preferably after the removal of cells/cellular debris) so as to provideenrichment in at least one miRNA species, and preferably a plurality ofmiRNA species. As used in the context of this disclosure, the term“enrichment” refers to the step of increasing the concentration of oneor more miRNA species in the acellular pro-tolerogenic preparation whencompared to conditioned pro-tolerogenic medium/blood. In an embodiment,the term enrichment refers to the step of increasing, in the acellularpro-tolerogenic preparation, the concentration but not the relativeabundance of the miRNA species present in the conditionedpro-tolerogenic medium/blood. In still another embodiment, theenrichment step can comprise substantially isolating the miRNA speciesfrom other components that may be present the conditionedpro-tolerogenic medium/blood (e.g., proteins such as cytokines forexample). This enrichment step can be completed using various methodsknown to those skilled in the art, for example, chromatography,precipitation, etc. Since most of the immuno-modulatory effects of theacellular pro-tolerogenic preparations reside in a fraction sensitive toribonucleic acid degradation (e.g., RNase degradation), this processstep should be conducted in conditions which would substantially limitor even inhibit ribonucleic acid degradation.

The conditioned pro-tolerogenic preparation can also be processed tosubstantially remove the protein components (including the cytokines)and/or the deoxyribonucleic acid components that may be present. Suchfurther purification step can be made, for example, by using proteinase(to provide a protein-free acellular pro-tolerogenic preparation), DNAse(to provide a DNA-free acellular pro-tolerogenic preparation),chromatography or filtration (to provide a fraction enriched insize-specific components present in the conditioned pro-tolerogenicmedium/blood).

In some embodiments, it is also contemplated that the acellularpro-tolerogenic preparation be submitted to the selective enrichment incomponents of the conditioned medium/blood having a relative size equalto or lower than about 20 kDa, 19 kDa, 18 kDa, 17 kDa, 16 kDa, 15 kDa,14 kDa, 13 kDa, 12 kDa, 11 kDa, 10 kDa, 9 kDa, 8 kDa, 7 kDa, 6 kDa, 5kDa, 4 kDa or 3 kDa.

Once the acellular pro-tolerogenic preparation has been obtained, it canbe formulated for administration to the subject. The formulation stepcan comprise admixing the acellular pro-tolerogenic preparations withpharmaceutically acceptable diluents, preservatives, solubilizers,emulsifiers, and/or carriers. The formulations are preferably in aliquid injectable form and can include diluents of various buffercontent (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength,additives such as albumin or gelatin to prevent absorption to surfaces.The formulations can comprise pharmaceutically acceptable solubilizingagents (e.g., glycerol, polyethylene glycerol), anti-oxidants (e.g.,ascorbic acid, sodium metabisulfite), preservatives (e.g., thimerosal,benzyl alcohol, parabens), bulking substances or tonicity modifiers(e.g., lactose, mannitol). The formulation step can also compriseplacing the acellular pro-tolerogenic preparation in a recipient (e.g.,physically distinct) which will prevent direct contact with theacellular pro-inflammatory preparation.

In some embodiments, the acellular pro-tolerogenic preparations can beformulated for simultaneous or sequential use with other substancescapable of favoring a state of immune tolerance, for example cortisone,IL-10, IL-11 and/or IL-12.

(ii) Process for Obtaining the Acellular Pro-Inflammatory Preparation

The acellular pro-inflammatory preparations presented described hereincan be obtained by contacting two distinct and allogeneic leukocytepopulations (referred herein to the third leukocyte and the fourthleukocyte). The two leukocyte populations are contacted under conditionsso as to allow (and in some embodiments to favor) pro-inflammatoryallo-recognition and to prevent (and in some embodiments to inhibit)pro-tolerogenic allo-recognition.

In some embodiments, the third and fourth leukocytes are used in aunmodified form (e.g., they are not modified to bear on their surface alow-immunogenic polymer). In alternative embodiments, it is possiblethat the third and/or the fourth leukocyte be modified to bear on theirsurface a low-immunogenic polymer. In such embodiment, it is importantthat the polymer grafted or the conditions used to graft the polymer donot significantly alter the ability of the two leukocyte populations tomediate a pro-inflammatory allo-recognition. It is important that thepolymer used exhibits both low-immunogenicity and biocompatibility onceintroduced into a cell culture system or administered to the testsubject. Polyethylene glycol (particularly methoxypoly(ethyleneglycol)), 2-alkyloxazoline (POZ) such as, for example,polyethyloxazoline (PEOZ) and hyperbranched polyglycerol (HPG) areexemplary polymers which all exhibit low immunogenicity andbiocompatibility and can be successfully used to modify the third and/orfourth leukocyte. In some embodiments, it is preferable to use a singletype of polymer to modify the surface of leukocytes. In otherembodiments, it is possible to use at least two distinct types ofpolymers to modify the surface of the leukocyte.

In an embodiment, the low-immunogenic biocompatible polymer can becovalently associated with the membrane-associated protein(s) of theleukocyte by creating a reactive site on the polymer (for example bydeprotecting a chemical group) and contacting the polymer with theleukocyte. For example, for covalently binding a methoxypoly(ethyleneglycol) to the surface of a leukocyte, it is possible to incubate amethoxypoly(-ethylene glycol) succinimidyl valerate (reactive polymer)in the presence of the leukocyte. The contact between the reactivepolymer and the leukocyte is performed under conditions sufficient forproviding a grafting density which will allow pro-inflammatoryallo-recognition and prevent pro-tolerogenic allo-recognition. In anembodiment, the polymer is grafted to a viable leukocyte and underconditions which will retain the viability of the leukocyte. A linker,positioned between the surface of the leukocyte and the polymer, canoptionally be used. Examples of such polymers and linkers are describedin U.S. Pat. Nos. 5,908,624; 8,007,784 and 8,067,151. In anotherembodiment, the low-immunogenic biocompatible polymer can be integratedwithin the lipid bilayer of the cytoplasmic membrane of the leukocyte byusing a lipid-modified polymer.

As indicated above, it is important that the low-immunogenicbiocompatible polymer be grafted at a density sufficient allowingpro-inflammatory allo-recognition while preventing pro-tolerogenicallo-recognition of the third leukocyte by the fourth leukocyte (andvice versa). In an embodiment, the polymer is polyethylene glycol (e.g.,linear) and has an average molecular weight between 2 and 40 KDa as wellas any combinations of molecular weight within this range. In anotherembodiment, the polymer is polyethylene glycol (e.g. linear) and has anaverage molecular weight between 2 and 40 KDa as well as anycombinations of molecular weight within this range. In a furtherembodiment, the average molecular weight of the PEG to be grafted is atleast 2, 3, 4, 5, 10, 15, 20, 25, 30, 35 or 40 kDa. In anotherembodiment, the average molecular weight of the PEG to be granted is nomore than 40, 35, 30, 25, 20, 15, 10, 5, 4, 3, or 2 kDa. In anotherembodiment, the grafting concentration of the polymer (per 20×10⁶ cells)is no more than 2.4, 2.0, 1.0, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.01 or0.005 mM. In still another embodiment, the grafting concentration of thepolymer (per 20×10⁶ cells) is equal to or lower than 0.005, 0.01, 0.05,0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 2.0, 2.4 mM. In embodiments where thepolymer is grafter to affect the viability of the leukocyte (for exampleby creating cellular instability, cellular fragmentation orvesiculization, the concentration of the polymer (per 20×10⁶ cells) isequal to or higher than 10 mM. In order to determine if pro-inflammatoryallo-recognition occurs, various techniques are known to those skilledin the art and include, but are not limited to, a standard mixedlymphocyte reaction (MLR), high molecular weight mitogen stimulation(e.g. PHA stimulation) as well as flow cytometry (Chen and Scott, 2006).In order to determine if a pro-tolerogenic allo-recognition occurs,various techniques are known to those skilled in the art and include,but are not limited to, the assessment of the level of expansion anddifferentiation of Treg cells and or prevention of Th17expansion/differentiation.

The third leukocyte can be optionally modified to refrain from beingproliferative. This modification preferably occurs prior to itsintroduction in a cell culture system or its administration into a testsubject. For example, the leukocyte can be irradiated (e.g.γ-irradiation) prior to its introduction in a cell culture system or inthe test subject. Upon irradiation, the leukocyte is not consideredviable (e.g. capable of proliferation). In an embodiment, polymergrafting can affect the leukocyte viability and be used to refrain theleukocyte from proliferating. Alternatively, leukocyte can be treatedwith a pharmacological agent capable of halting cell cycle progression.Upon the administration of such pharmacological agent, the leukocyte isconsidered viable since it can resume cellular proliferation when theagent is removed from the cell-containing medium.

It is also contemplated that the fourth leukocyte (which can optionallybe modified with the low-immunogenic and biocompatible polymer) be alsooptionally modified to refrain from being proliferative. For example,the leukocyte can be irradiated (e.g. γ-irradiation) prior to itsintroduction in a cell culture system or in the test subject. Uponirradiation, the leukocyte is not considered viable (e.g. capable ofproliferation). In an embodiment, polymer grafting can affect theleukocyte viability and can be used to refrain the leukocyte fromproliferating. Alternatively, leukocyte can be treated with apharmacological agent capable of halting cell cycle progression. Uponthe administration of such pharmacological agent, the leukocyte isconsidered viable since it can resume cellular proliferation when theagent is removed from the cell-containing medium. However, when thefourth leukocyte is modified from being proliferative, it is importantthe third leukocyte with which it is being contacted remainsproliferative.

In order to generate the acellular pro-inflammatory preparations, it isnot necessary to provide homogeneous leukocyte populations. For example,the third leukocyte population (such as, for example a PBMCs orsplenocytes) can be introduced in a cell culture system and contactedwith a fourth leukocyte population (such as, for example a PBMCs orsplenocytes) or administered to the test subject. However, in someembodiments, it is possible to provide and contact more homogeneousleukocyte populations. For example, the third leukocyte population canbe relatively homogenous (such as, for example, a T cell population) andintroduced in a cell culture system comprising a fourth leukocytepopulation (such as, for example a PBMC or splenocyte) or administeredto the test subject. In another example, the third leukocyte population(such as, for example a PBMC or splenocyte) can be introduced in a cellculture system comprising a fourth leukocyte population which can berelatively homogeneous (such as, for example, a T cell population). In afurther example, the third leukocyte population can be relativelyhomogenous (such as, for example, a T cell population) and introduced ina cell culture system comprising a fourth leukocyte population which canbe relatively homogeneous (such as, for example, a T cell population).

To provide the acellular pro-inflammatory preparations describedherewith, the leukocytes used can be mature leukocytes or be provided inthe form of stem cells (e.g., for example non-embryonic stem cells). Forexample, leukocytes can be obtained from isolating peripheral bloodmononuclear cells (PBMC) from the subject. Optionally, the PBMCs can bedifferentiated in vitro into dendritic (DC) or DC-like cells.Alternatively, the leukocytes can be obtained from the spleen (e.g.splenocytes). Leukocytes usually include T cells, B cells and antigenpresenting cells. In some embodiments, cells of sufficient antigenicvariation and immunogenicity are used. In addition, for providing theacellular pro-inflammatory preparations, the leukocytes but noterythrocytes are necessary since the polymer-modified erythrocytes arenot capable of eliciting a pro-inflammatory allo-recognition whenadministered in a test subject. However, traces of erythrocytes in theleukocyte population used are tolerated (for example, less than about10%, less than about 5% or less than about 1% of the total number ofcells in the preparation).

Even though it is not necessary to further purify the leukocytes toprovide the acellular pro-inflammatory preparations, it is possible touse a pure cell population or a relatively homogenous population ofcells as leukocytes. This “pure” cell population and “relativehomogenous population” of cells can, for example, essentially consistessentially of a single cell type of T cells, B cells, antigenpresenting cells (APC) or stem cells. Alternatively, the population ofcells can consist essentially of more than one cell type. The populationof cells can be obtained through conventional methods (for example cellsorting or magnetic beads). In an embodiment, when the population ofcells consist of a single cell type (for example, T cells), thepercentage of the cell type with respect to the total population ofcells is at least 90%, at least 95% or at least 99%. The relativelyhomogenous population of cells is expected to contain some contaminatingcells, for example less than 10%, less than 5% or less than 1% of thetotal population of cells.

The third leukocyte and/or fourth leukocyte can be obtained from anyanimals, but are preferably derived from mammals (such as, for example,humans and mice). In an embodiment, the third or fourth leukocyte can beobtained from a subject intended to be treated with the acellularpro-inflammatory preparation.

The third and/or fourth leukocyte can be expanded in vitro prior to theintroduction in a cell culture system or the administration to a testsubject.

As indicated above, the third and fourth leukocyte are contacted underconditions to allow pro-inflammatory allo-recognition (e.g. expansion ofpro-inflammatory T cells and/or differentiation of naïve T cells inpro-inflammatory T cells) and prevent/inhibit pro-tolerogenicallo-recognition (e.g. expansion of Treg cells and/or differentiation ofnaïve T cells in Treg cells). When the contact occurs in vitro, it isimportant that the third leukocyte and the fourth leukocyte be culturedunder conditions allowing physical contact between the two leukocytepopulations and for a time sufficient to provide a conditionedpro-inflammatory medium. As used herein, a conditioned pro-inflammatorymedium refers to physical components of a cell culture (or fractionthereof, such as the cell culture supernatant) obtained by contactingthe third and the fourth leukocyte and having the pro-inflammatoryproperties described herein. Usually, the conditioned medium is obtainedat least 24 hours after the initial contact between the third and fourthleukocyte. In some embodiment, the conditioned medium is obtained atleast 48 hours or at least 72 hours after the initial contact betweenthe third and the fourth leukocyte. In an embodiment, the conditionedmedium can be obtained after at least 24 hours of incubating a thirdleukocyte with a fourth leukocyte. When the incubation takes place in a24-well plate, the concentration of each leukocyte population can be,for example, at least 1×10⁶ cells.

When the contact occurs in vivo, it is important that the thirdleukocyte be administered to an immune competent test subject (bearingthe fourth leukocyte) and that the blood or blood fraction be obtainedat a later a time sufficient to provide a conditioned pro-inflammatoryblood. The test subject is a subject being immune competent and having aTreg/pro-inflammatory ratio which is substantially similar to age- andsex-matched healthy subjects. As used herein, the conditionedpro-inflammatory blood refers to physical components present in theblood (or fraction thereof, such as the plasma) obtained byadministering the third leukocyte to the immune competent test subjectand having the pro-inflammatory properties described herein. It isrecognized by those skilled in the art that the conditionedpro-inflammatory blood may be obtained more rapidly by increasing theamount of leukocytes being administered or administering more than once(for example one, twice or thrice) the third leukocyte. Usually, theconditioned pro-inflammatory blood can be obtained at least one dayafter the administration of the third leukocyte. In some embodiment, theconditioned pro-inflammatory blood is obtained at least 2, 3, 4, 5, 6 or7 days after the administration of the third leukocyte. In anembodiment, the conditioned pro-inflammatory blood can be obtained byadministering at least 5×10⁶ allogeneic leukocytes to the test subject(e.g., test mouse) and recuperating the plasma five days later. In someembodiments, the conditioned pro-inflammatory blood can be obtained byadministering at least 20×10⁶ allogeneic leukocytes to the test subject(e.g., test mouse).

In an embodiment, in order to obtain the conditioned pro-inflammatoryblood, the test subject is transfused in conditions so as to allow apro-inflammatory allo-recognition but to prevent the onset of GVHD. Thethird leukocyte is considered immunogenic (e.g. allogeneic) with respectto the test subject because when the third leukocyte is transfused intothe animal, an immune response (e.g. a cell-mediated immune response,preferably a pro-inflammatory allo-recognition) occurs. In anotherembodiment, the third leukocyte can be xenogeneic with respect to thetest subject. However, the third leukocyte cannot be autologous orsyngeneic to the animal. In some embodiments, the third leukocyte can beallogeneic or xenogeneic to the subject which will be treated with theconditioned pro-inflammatory blood. In alternative embodiment, the thirdleukocyte can be syngeneic or derived from the subject which will betreated with the conditioned pro-inflammatory blood. In an embodiment,the third allogeneic leukocyte can be modified to bear on its surface apolymer. However, as indicated above, the polymer, when present, must beselected or grafted at a density so as to allow the pro-inflammatoryallo-recognition of the first leukocyte by the recipient. When the thirdleukocyte is modified to bear on its surface a polymer, it can bemodified to be non-proliferative either prior to or after the polymermodification.

As indicated herein, the two leukocyte populations are consideredallogeneic (and in some embodiments, xenogeneic). When the acellularpro-inflammatory preparation is obtained in vivo by, for example, aconditioned blood/blood fraction can be obtained by administering thethird leukocyte to the test subject, the third leukocyte can beallogeneic or xenogeneic to the test subject. In such embodiment, it isalso contemplated that the third leukocyte be autologous, syngeneic,allogeneic or xenogeneic to a treated subject who is going to receivethe acellular pro-inflammatory preparation. When the acellularpro-inflammatory preparation is obtained in vitro by, for example, aconditioned medium can be obtained by co-culturing the third leukocytewith the fourth leukocyte, the third leukocyte can be allogeneic orxenogeneic to the fourth leukocyte. In such embodiment, it is alsocontemplated that the third leukocyte be autologous, syngeneic,allogeneic or xenogeneic to a treated subject who is going to receivethe acellular pro-inflammatory preparation. In addition, it is alsocontemplated that the fourth leukocyte be autologous, syngeneic,allogeneic or xenogeneic to a treated subject who is going to receivethe acellular pro-inflammatory preparation.

Once the conditioned pro-inflammatory preparation has been obtained, itcan be further processed to substantially remove the cells and cellulardebris that can be present. This processing step can be achieved bysubmitting the conditioned pro-inflammatory medium or the conditionedpro-inflammatory blood to a centrifugation step and/or a filtrationstep. For example, blood can be processed as to obtain the plasma. Sincethe majority of the immuno-modulatory effects of the acellularpro-inflammatory preparations reside in a fraction sensitive toribonucleic acid degradation (e.g., RNase degradation), this processstep should be conducted in conditions which would substantially limitor even inhibit ribonucleic acid degradation.

The conditioned pro-inflammatory preparation can also be processed(preferably after the removal of cells/cellular debris) so as to provideenrichment in at least one miRNA species, and preferably a plurality ofmiRNA species. As used in the context of this disclosure, the term“enrichment” refers to the step of increasing the concentration of oneor more miRNA species in the acellular pro-inflammatory preparation whencompared to conditioned medium/blood. In an embodiment, the termenrichment refers to the step of increasing, in the acellularpro-inflammatory preparation, the concentration but not the relativeabundance of the miRNA species present in the conditionedpro-inflammatory medium/blood. In still another embodiment, theenrichment step can comprise substantially isolating the miRNA speciesfrom other components that may be present the conditionedpro-inflammatory medium/blood (e.g., proteins such as cytokines forexample). This enrichment step can be completed using various methodsknown to those skilled in the art, for example, chromatography,precipitation, etc. Since most of the immuno-modulatory effects of theacellular pro-inflammatory preparations reside in a fraction sensitiveto ribonucleic acid degradation (e.g. RNase degradation), this processstep should be conducted in conditions which would substantially limitor even inhibit ribonucleic acid degradation.

The conditioned pro-inflammatory preparation can also be processed tosubstantially remove the protein components (including the cytokines)and/or the deoxyribonucleic acid components that may be present. Suchfurther purification step can be made, for example, by using proteinase(to provide a protein-free acellular pro-inflammatory preparation),DNAse (to provide a DNA-free acellular pro-inflammatory preparation),chromatography or filtration (to provide a fraction enriched insize-specific components present in the conditioned pro-inflammatorymedium/blood).

In some embodiments, it is also contemplated that the acellularpro-inflammatory preparation be submitted to the selective enrichment incomponents of the conditioned medium/blood having a relative size equalto or lower than about 20 kDa, 19 kDa, 18 kDa, 17 kDa, 16 kDa, 15 kDa,14 kDa, 13 kDa, 12 kDa, 11 kDa, 10 kDa, 9 kDa, 8 kDa, 7 kDa, 6 kDa, 5kDa, 4 kDa or 3 kDa.

Once the acellular pro-inflammatory preparation has been obtained, itcan be formulated for administration to the subject. The formulationstep can comprise admixing the acellular pro-inflammatory preparationwith pharmaceutically acceptable diluents, preservatives, solubilizers,emulsifiers, and/or carriers. The formulations are preferably in aliquid injectable form and can include diluents of various buffercontent (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength,additives such as albumin or gelatin to prevent absorption to surfaces.The formulations can comprise pharmaceutically acceptable solubilizingagents (e.g., glycerol, polyethylene glycerol), anti-oxidants (e.g.,ascorbic acid, sodium metabisulfite), preservatives (e.g., thimerosal,benzyl alcohol, parabens), bulking substances or tonicity modifiers(e.g., lactose, mannitol). The formulation step can also compriseplacing the acellular pro-inflammatory preparation in a recipient (e.g.,physically distinct) which will prevent direct contact with theacellular pro-tolerogenic preparation.

In addition, if the acellular pro-inflammatory preparation is destinedto be used to prevent, treat or alleviate the symptoms of cancer, it canbe formulated to be co-administered with an anti-neoplastic agent. Theacellular pro-inflammatory preparation can be formulated forsimultaneous administration with the anti-neoplastic agent by admixingthe anti-neoplastic agent with the acellular pro-inflammatorypreparation. Alternatively, the acellular pro-inflammatory preparationcan be formulated for administration prior to or after theanti-neoplastic agent, for example in a formulation that is physicallydistinct from the anti-neoplastic agent.

In another embodiment, if the acellular pro-inflammatory preparation isdestined to be used to prevent, treat or alleviate the symptoms of aninfection, it can be formulated to be co-administered with ananti-infective agent (such as an anti-parasitic, antibacterial,anti-viral or anti-fungal agent). The acellular pro-inflammatorypreparation can be formulated for simultaneous administration with theanti-infective agent by admixing the anti-infective agent with theacellular pro-inflammatory preparation. Alternatively, the acellularpro-inflammatory preparation can be formulated for administration priorto or after the anti-infective agent, for example in a formulation thatis physically distinct from the anti-infective agent.

In still another embodiment, if the acellular pro-inflammatorypreparation is destined to be used to allow a robust immune response toa vaccine, it can be formulated to be co-administered with the vaccine.The acellular pro-inflammatory preparation can be formulated forsimultaneous administration with the vaccine by admixing the vaccinewith the acellular pro-inflammatory preparation. Alternatively, theacellular pro-inflammatory preparation can be formulated foradministration prior to or after the vaccine, for example in aformulation that is physically distinct from the vaccine.

In yet another embodiment, the acellular pro-inflammatory preparationcan be formulated with other therapeutic agents capable of providingpro-inflammatory effects such as, for example IL-2, IL-4, TNF-α and/orINF-γ.

(iii) Characterization of the miRNA Fraction of the AcellularPreparations

As shown herein, the miRNA fraction of the acellular preparation isassociated with the majority of the immunomodulatory effects of theconditioned medium/blood. As also shown herein, the immunomodulatoryeffects of the miRNA fraction of the acellular preparations are greatlyreduced (and even abolished) when the components of the conditionedblood/medium having an average molecular weight lower than about 10 kDaare removed or upon treatment with a ribonucleic acid degradation agent(such as RNase A).

The acellular preparations described herein does comprise a plurality(also referred to a population) of distinct miRNA species whose relativeabundance differs between a conditioned pro-tolerogenic medium obtained,for example, from a mPEG MLR (e.g. in which two allogeneic leukocytepopulations are co-cultured under conditions so as to allowpro-tolerogenic allo-recognition), a conditioned pro-inflammatory mediumobtained, for example, from a control MLR (e.g. in which the twoallogeneic leukocyte populations are co-cultured under conditions so asto allow pro-inflammatory allo-recognition) or a control mediumobtained, for example, from resting cells (e.g. a single type ofcultured leukocyte population). The relative abundance of the miRNApopulation also differs between a conditioned pro-tolerogenic bloodobtained from the administration of polymer-modified allogeneic cells ina test subject (in which a pro-tolerogenic allo-recognition occurred), aconditioned blood obtained from the administration of unmodifiedallogeneic cells to a test subject (in which a pro-inflammatoryallo-recognition occurred) or a control blood obtained from naïve testsubject or sham-treated subjects. This modulation in the relativeabundance of the various miRNA species of the acellular preparation isbelieved to be tied to its immunomodulatory effects. The increasedabundance of single miRNA species, unchanged abundance of single miRNAspecies and/or decreased abundance of single miRNA species are believedto contribute to the immunomodulatory effects of the acellularpreparations. In an embodiment, in the acellular preparations, therelative pattern of expression of the miRNA species is conserved.

In an embodiment, the acellular (protolerogenic and/or pro-inflammatory)preparation comprises at least one miRNA species presented in FIG. 9. Inanother embodiment, the acellular preparation comprises any combinationof at least 2, 3, 4, 5, 10, 15, 20, 25, 30, 30, 35, 40, 45, 50, 55, 60,65, 70, 75 or 80 of the miRNA species presented in FIG. 9. In stillanother embodiment, the acellular preparation comprises all the miRNAspecies presented in FIG. 9. In yet another embodiment, the relativeabundance of the miRNA species in the acellular preparation is similarto the relative abundance of the miRNA species show in FIG. 9. FIG. 9provides the following miRNA species: hsa-let-7a-5p, hsa-let-7c,hsa-let-7d-5p, hsa-let-7e-5p, hsa-let-7g-5p, hsa-miR-103a-3p,hsa-miR-105-5p, hsa-miR-125a-5p, hsa-miR-125b-5p, hsa-miR-126-3p,hsa-miR-128, hsa-miR-130a-3p, hsa-miR-132-3p, hsa-miR-134,hsa-miR-135a-5p, hsa-miR-135b-5p, hsa-miR-138-5p, hsa-miR-142-3p,hsa-miR-142-5p, hsa-miR-143-3p, hsa-miR-145-5p, hsa-miR-146a-5p,hsa-miR-147a, hsa-miR-148a-3p, hsa-miR-149-5p, hsa-miR-150-5p,hsa-miR-152, hsa-miR-155-5p, hsa-miR-15a-5p, hsa-miR-15b-5p,hsa-miR-16-5p, hsa-miR-17-5p, hsa-miR-181a-5p, hsa-miR-182-5p,hsa-miR-183-5p, hsa-miR-184, hsa-miR-185-5p, hsa-miR-186-5p,hsa-miR-187-3p, hsa-miR-18a-5p, hsa-miR-18b-5p, hsa-miR-191-5p,hsa-miR-194-5p, hsa-miR-195-5p, hsa-miR-196a-5p, hsa-miR-19a-3p,hsa-miR-19b-3p, hsa-miR-200a-3p, hsa-miR-203a, hsa-miR-205-5p,hsa-miR-206, hsa-miR-20a-5p, hsa-miR-20b-5p, hsa-miR-21-5p, hsa-miR-210,hsa-miR-214-3p, hsa-miR-223-3p, hsa-miR-23b-3p, hsa-miR-26a-5p,hsa-miR-26b-5p, hsa-miR-27a-3p, hsa-miR-27b-3p, hsa-miR-298,hsa-miR-299-3p, hsa-miR-29b-3p, hsa-miR-29c-3p, hsa-miR-302a-3p,hsa-miR-30b-5p, hsa-miR-30c-5p, hsa-miR-30e-5p, hsa-miR-31-5p,hsa-miR-325, hsa-miR-335-5p, hsa-miR-34a-5p, hsa-miR-363-3p,hsa-miR-379-5p, hsa-miR-383, hsa-miR-409-3p, hsa-miR-451a,hsa-miR-493-3p, hsa-miR-574-3p, hsa-miR-9-5p, hsa-miR-98-5p andhsa-miR-99b-5p.

In another embodiment, the acellular (protolerogenic and/orpro-inflammatory) prepration comprises at least one miRNA species whoserelative abundance is increased in the conditioned medium/blood obtainedfrom using non-modified leukocytes (capable of allowing apro-inflammatory allo-recognition) when compared to a conditionedmedium/blood obtained from using polymer-modified leukocytes (capable ofallowing a pro-tolerogenic allo-recognition) or resting cells/naïveblood. Such miRNA species are listed in Tables 1A to 1D. In anembodiment, the relative abundance of miRNA species in the acellularpreparations is similar to the relative abundance of miRNA specieslisted in any one of Tables 1A to 1D.

TABLE 1A miRNA species in which the relative abundance in theconditioned medium of the control MLR is increased when compared to themiRNA species's conditioned medium from resting cells/naïve blood (asdetermined in FIG. 9). hsa-let-7c hsa-miR-105-5p hsa-miR-130a-3phsa-miR-134 hsa-miR-135a-5p hsa-miR-135b-5p* hsa-miR-142-3phsa-miR-142-5p hsa-miR-147a* hsa-miR-149-5p hsa-miR-155-5p*hsa-miR-15a-5p hsa-miR-181a-5p hsa-miR-183-5p* hsa-miR-187-3phsa-miR-18a-5p hsa-miR-18b-5p hsa-miR-200a-3p hsa-miR-203a*hsa-miR-205-5p hsa-miR-206* hsa-miR-210 hsa-miR-214-3p* hsa-miR-299-3phsa-miR-29b-3p hsa-miR-302a-3p* hsa-miR-31-5p hsa-miR-325*hsa-miR-363-3p* hsa-miR-383 hsa-miR-451a hsa-miR-493-3p hsa-miR-574-3phsa-miR-9-5p* miRNA species identified with an * show a log₂ foldregulation change or a p ≤ 0.05 on a volcano plot.

In a further embodiment, the acellular (protolerogenic and/orpro-inflammatory) preparation comprises at least one miRNA specieslisted Table 1A. In still a further embodiment, the acellular(protolerogenic and/or pro-inflammatory) preparation comprises acombination of at least 2, 3, 4, 5, 10, 15, 20, 25, 30 or 33 of any oneof the miRNA species listed in Table 1A. In yet a further embodiment,the acellular (protolerogenic and/or pro-inflammatory) preparationcomprises all the miRNA species listed in Table 1A.

In an embodiment, the acellular (protolerogenic and/or pro-inflammatory)preparation comprises at least one (or any combination of) miRNA specieslisted in Table 1A and showing a log₂ fold regulation change or a p≤0.05on a volcano plot (e.g., hsa-miR-135b-5p, hsa-miR-147a, hsa-miR-155-5p,hsa-miR-183-5p, hsa-miR-203a, hsa-miR-206, hsa-miR-214-3p,hsa-miR-302a-3p, hsa-miR-325, hsa-miR-363-3p, hsa-miR-9-5p).

TABLE 1B miRNA species in which the relative abundance in theconditioned medium of control MRL is increased when compared to themiRNA species' abundance in the conditioned medium of the mPEG MRL andis decreased when compared the miRNA species' abundance in the mediumfrom resting cells/naïve blood (as determined in FIG. 9).hsa-miR-183-5p* hsa-miR-203a* hsa-miR-363-3p* miRNA species identifiedwith an * show a log₂ fold regulation change or a p ≤ 0.05 on a volcanoplot.

In a further embodiment, the acellular (protolerogenic and/orpro-inflammatory) preparation comprises at least one miRNA specieslisted Table 1B. In still a further embodiment, the acellular(protolerogenic and/or pro-inflammatory) preparation comprises acombination of at least 2 of any one of the miRNA species listed inTable 1B. In yet a further embodiment, the acellular (protolerogenicand/or pro-inflammatory) preparation comprises all the miRNA specieslisted in Table 1B.

TABLE 1C miRNA species in which the relative abundance in theconditioned medium of the control MLR is increased when compared tomiRNA species' abundance in the conditioned medium of the restingcells/naïve blood. The relative abundance of these miRNA species is alsoincreased in the conditioned medium of the mPEG MLR when compared tomiRNA species' abundance in the conditioned medium of the restingcells/naï ve blood (as determined in FIG. 9). hsa-let-7c hsa-miR-105-5phsa-miR-130a-3p hsa-miR-134 hsa-miR-135a-5p hsa-miR-135b-5p*hsa-miR-142-3p hsa-miR-142-5p hsa-miR-147a* hsa-miR-149-5p*hsa-miR-155-5p* hsa-miR-15a-5p hsa-miR-181a-5p hsa-miR-187-3phsa-miR-18a-5p hsa-miR-18b-5p hsa-miR-200a-3p hsa-miR-205-5phsa-miR-206* hsa-miR-210 hsa-miR-214-3p* hsa-miR-299-3p hsa-miR-29b-3phsa-miR-302a-3p* hsa-miR-31-5p hsa-miR-383 hsa-miR-451a hsa-miR-493-3phsa-miR-574-3p hsa-miR-9-5p* miRNA species identified with an * show alog₂ fold regulation change or a p ≤ 0.05 on a volcano plot.

In a further embodiment, the acellular (protolerogenic and/orpro-inflammatory) preparation comprises at least one miRNA specieslisted Table 10. In still a further embodiment, the acellular(protolerogenic and/or pro-inflammatory) preparation comprises acombination of at least 2, 3, 4, 5, 10, 15, 20, 25 or 30 of any one ofmiRNA species listed in Table 10. In yet a further embodiment, theacellular (protolerogenic and/or pro-inflammatory) preparation comprisesall the miRNA species listed in Table 10.

In an embodiment, the acellular (protolerogenic and/or pro-inflammatory)preparation comprises at least one (or any combination of) miRNA specieslisted in Table 10 and showing a log₂ fold regulation change or a p≤0.05on a volcano plot (e.g., hsa-miR-135b-5p, hsa-miR-147a, hsa-miR-149-5p,hsa-miR-155-5p, hsa-miR-206, hsa-miR-214-3p, hsa-miR-302a-3p and/orhsa-miR-9-5p). In an embodiment, the acellular (protolerogenic and/orpro-inflammatory) preparation comprises at least one of hsa-mir-147a andhsa-mir-9-5p.

TABLE 1D Selection of the miRNA species from Table 1C which show anincrease of a log₂ fold regulation change or a p ≤ 0.05 on a volcanoplot. hsa-miR-135b-5p hsa-miR-147a hsa-miR-149-5p hsa-miR-155-5phsa-miR-183-5p hsa-miR-203a-5p hsa-miR-206 hsa-miR-214-3phsa-miR-302a-3p hsa-miR-363-3p hsa-miR-9-5p

In a further embodiment, the acellular (protolerogenic and/orpro-inflammatory) preparation comprises at least one miRNA specieslisted Table 1D. In still a further embodiment, the acellular(protolerogenic and/or pro-inflammatory) preparation comprises acombination of at least 2, 3, 4, 5, 6 or 7 of any one of miRNA specieslisted in Table 1D. In yet a further embodiment, the acellular(protolerogenic and/or pro-inflammatory) preparation comprises all themiRNA species listed in Table 1D.

In another embodiment, the acellular (protolerogenic and/orpro-inflammatory) preparation comprises at least one miRNA species whoserelative abundance is decreased when compared to a conditionedmedium/blood obtained from using polymer-modified leukocytes (capable ofallowing pro-tolerogenic allo-recognition) or the medium from restingcells/naïve blood. Such miRNA species are listed in Tables 2A to 2D. Inanother embodiment, the relative abundance of the miRNA species in theacellular preparations is similar to the relative abundance of the miRNAspecies presented in any one of Tables 2A to 2D.

TABLE 2A miRNA species in which the relative abundance in theconditioned medium of the control MLR is decreased when compared to themiRNA species's conditioned medium from resting cells/naïve blood (asdetermined in FIG. 9). hsa-let-7a-5p* Has-let-7d-5p hsa-let-7e-5p*hsa-let-7g-5p hsa-miR-103a-3p hsa-miR-125a-5p hsa-miR-125b-5phsa-miR-126-3p hsa-miR-128 hsa-miR-132-3p* hsa-miR-138-5p hsa-miR-143-3phsa-miR-145-5p hsa-miR-146a-5p hsa-miR-148a-3p hsa-miR-150-5phsa-miR-152 hsa-miR-15b-5p hsa-miR-16-5p hsa-miR-17-5p hsa-miR-182-5phsa-miR-184 hsa-miR-185-5p hsa-miR-186-5p has-miR-191-5p hsa-miR-194-5phsa-miR-195-5p hsa-miR-196a-5p hsa-miR-19a-3p hsa-miR-19b-3phsa-miR-20a-5p hsa-miR-20b-5p hsa-miR-21-5p* hsa-miR-223-3phsa-miR-23b-3p hsa-miR-26a-5p hsa-miR-26b-5p hsa-miR-27a-3p*hsa-miR-27b-3p* hsa-miR-298* hsa-miR-29c-3p hsa-miR-30b-5phsa-miR-30c-5p hsa-miR-30e-5p hsa-miR-335-5p hsa-miR-34a-5p*hsa-miR-379-5p hsa-miR-409-3p hsa-miR-98-5p hsa-miR-99b-5p miRNA speciesidentified with an * show a log2 fold regulation change or a p ≤ 0.05 ona volcano plot.

In a further embodiment, the acellular (protolerogenic and/orpro-inflammatory) preparation comprises at least one miRNA specieslisted Table 2A. In still a further embodiment, the acellular(protolerogenic and/or pro-inflammatory) preparation comprises acombination of at least 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 46of any one of miRNA species listed in Table 2A. In yet a furtherembodiment, the acellular (protolerogenic and/or pro-inflammatory)preparation comprises all the miRNA species listed in Table 2A.

In an embodiment, the acellular (protolerogenic and/or pro-inflammatory)preparation comprises at least one (or any combination of) miRNA specieslisted in Table 2A and showing a log₂ fold regulation change or a p≤0.05on a volcano plot (e.g., hsa-let-7a-5p, hsa-let-7e-5p, hsa-miR-132-3p,hsa-miR-21-5p, hsa-miR-27a-3p, hsa-miR-27b-3p, hsa-miR-298,hsa-miR-34a-5p).

TABLE 2B miRNA species in which the relative abundance in theconditioned medium of control MRL is decreased when compared to themiRNA species' abundance in the conditioned medium of the mPEG MRL (asdetermined in FIG. 9). hsa-let-7a-5p* hsa-let-7e-5p* hsa-miR-132-3p*hsa-miR-21-5p* hsa-miR-27a-3p* hsa-miR-27b-3p* hsa-miR-298*hsa-miR-34a-5p* miRNA species identified with an * show a log₂ foldregulation change.

In a further embodiment, the acellular (protolerogenic and/orpro-inflammatory) preparation comprises at least one miRNA specieslisted Table 2B. In still a further embodiment, the acellular(protolerogenic and/or pro-inflammatory) preparation comprises acombination of at least 2, 3, 4, 5, 6 or 7 of any one of the miRNAspecies listed in Table 2B. In yet a further embodiment, the acellular(protolerogenic and/or pro-inflammatory) preparation comprises all themiRNA species listed in Table 2B.

TABLE 2C Selection of the miRNA species from Table 2B which show a log₂fold regulation change or a p ≤ 0.05 on a volcano plot. hsa-let-7a-5phsa-let-7e-5p hsa-miR-132-3p hsa-miR-21-5p hsa-miR-27a-3p hsa-miR-27b-3phsa-miR-298 hsa-miR-34a-5p

In a further embodiment, the acellular (protolerogenic and/orpro-inflammatory) preparation comprises at least one miRNA specieslisted Table 2C. In still a further embodiment, the acellular(protolerogenic and/or pro-inflammatory) preparation comprises acombination of at least 2, 3, 4, 5, 6 or 7 any one of miRNA specieslisted in Table 2C. In yet a further embodiment, the acellular(protolerogenic and/or pro-inflammatory) preparation comprises all themiRNA species listed in Table 2C.

TABLE 2D miRNA species in which the relative abundance in theconditioned medium of control MRL and in the condition medium of themPRG MLR is decreased when compared to the miRNA species' abundance inthe conditioned medium from resting cells/naïve blood (as determined inFIG. 9). hsa-let-7d-5p hsa-let-7g-5p hsa-miR-103a-3p hsa-miR-125a-5phsa-miR-125b-5p hsa-miR-126-3p hsa-miR-128 hsa-miR-138-5p hsa-miR-143-3phsa-miR-145-5p hsa-miR-146a-5p hsa-miR-148a-3p hsa-miR-150-5phsa-miR-152 hsa-miR-15b-5p hsa-miR-16-5p hsa-miR-17-5p hsa-miR-182-5phsa-miR-184 hsa-miR-185-5p hsa-miR-186-5p has-miR-191-5p hsa-miR-194-5phsa-miR-195-5p hsa-miR-196a-5p hsa-miR-19a-3p hsa-miR-19b-3phsa-miR-20a-5p hsa-miR-20b-5p hsa-miR-223-3p hsa-miR-23b-3phsa-miR-26a-5p hsa-miR-26b-5p hsa-miR-29c-3p hsa-miR-30b-5phsa-miR-30c-5p hsa-miR-30e-5p hsa-miR-335-5p hsa-miR-379-5phsa-miR-409-3p hsa-miR-98-5p hsa-miR-99b-5p

In a further embodiment, the acellular (protolerogenic and/orpro-inflammatory) preparation comprises at least one miRNA specieslisted Table 2D. In still a further embodiment, the acellular(protolerogenic and/or pro-inflammatory) preparation comprises acombination of at least 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40 or 42 ofany one of miRNA species listed in Table 2D. In yet a furtherembodiment, the acellular (protolerogenic and/or pro-inflammatory)preparation comprises all the miRNA species listed in Table 2D.

It is contemplated that the acellular (protolerogenic and/orpro-inflammatory) preparation comprises at least one (and in anembodiment any combination of) miRNAs species from any one of Tables 1Ato 1D and at least one (and in an embodiment any combination of) miRNAsspecies from any one of Tables 2A to 2D.

In yet another embodiment, the acellular (protolerogenic and/orpro-inflammatory) preparation can comprise at least one of the miRNAspecies identified in the volcano plots of FIG. 8. In still anotherembodiment, the acellular (protolerogenic and/or pro-inflammatory)preparation comprises at least one (or any combination of) miRNA speciespresented on FIG. 8A which exhibits at least a log₂ fold modulation inabundance (e.g. miR-302a-3p, miR214-3p, miR-147a, miR206, miR 155-5pand/or miR-9-5p). In yet still another embodiment, the acellular(protolerogenic and/or pro-inflammatory) preparation comprises at leastone (or any combination of) of miRNA species presented on FIG. 8A whichexhibits at least p≤0.05 (e.g. miR214-3p, miR-147a, miR206, miR 155-5pand/or miR-9-5p). In yet another embodiment, the acellular(protolerogenic and/or pro-inflammatory) preparation comprises at leastone (or any combination of) miRNA species presented on FIG. 8B whichexhibits at least a log₂ fold modulation in abundance (e.g. miR-149-5pand/or miR-214-3p). In yet still another embodiment, the acellular(protolerogenic and/or pro-inflammatory) preparation comprises the miRNAspecies presented on FIG. 8B which exhibits at least p≤0.05 (e.g.miR-214-3p). In yet another embodiment, the acellular (protolerogenicand/or pro-inflammatory) preparation comprises at least one (or anycombination of) miRNA species presented on FIG. 8C which exhibits atleast a log₂ fold modulation in abundance (e.g. miR-147a, miR-183-5p,miR-9-5p and/or miR-155-5p). In yet still another embodiment, theacellular (protolerogenic and/or pro-inflammatory) preparation comprisesat least one (or any combination of) miRNA species presented on FIG. 8Cwhich exhibits at least p≤0.05 (e.g. miR-9-5p and/or miR-155-5p). Inanother embodiment, the miRNA species that are present in the acellularpreparations have a relative abundance which is similar to thosepresented in any one of FIGS. 8A to 8C.

It is contemplated that the acellular (protolerogenic and/orpro-inflammatory) preparation comprises at least one (and in anembodiment any combination of) miRNAs species from any one of Tables 1Ato 1D, at least one (and in an embodiment any combination of) miRNAsspecies from any one of Tables 2A to 2D and at least one (and in anembodiment any combination of) miRNA species identified in any one ofthe FIGS. 8A to 8C.

Methods for Modulating the Treg/Pro-Inflammatory T Cells Ratio

The present disclosure provides methods and associated therapeutic usesfor modulating the ratio of the level of regulatory T cells with respectto the level of pro-inflammatory T cells during a period of time for anindividual. In the present context, the term modulation refers to asequential increase in the immune response (e.g., a reduction in theTregs/pro-inflammatory T cell ratio) and decrease in the immune response(e.g., an increase in the Tregs/pro-inflammatory T cell ratio) of asubject. In the present context, the increase can precede the decreaseor the decrease can precede the increase. This modulation in immuneresponse is an intentional one since it is triggered/induced by theadministration of an exogenous biological acellular preparation.

In a first embodiment, the method allows to modulation of the ratio ofTreg to pro-inflammatory T cells in a subject having received a firsttherapeutic dose (or multiple doses) of an acellular pro-tolerogenicpreparation. The method thus allows, in such subject, a decrease in theratio of Treg to pro-inflammatory T cells which was previouslyintentionally increased (by the administration of one or more doses ofthe acellular pro-tolerogenic preparation). In order to achieve thisreduction in the ratio of Treg to pro-inflammatory T cells, at least onedose (or multiple doses) of the acellular pro-inflammatory preparationis administered to the subject. Further modulation of the ratio can beachieved by administering sequentially and, optionally in an alternatefashion, additional therapeutic doses of the acellular pro-tolerogenicpreparations and the acellular pro-inflammatory preparations. In someembodiments, it may be advantageous to determine, prior to theadministration of the first or any therapeutic dose of the acellularpro-inflammatory preparation, if the subject is in need of decreasingits Tregs to pro-inflammatory T cells ratio. This can be done byactually characterizing T cell subpopulations in the subject intended tobe treated, measuring such ratio (and comparing it to ratios associatedwith age- and sex-matched healthy subjects) and/or identifying acondition which would require immune stimulation (e.g., an infection ora vaccine for example). The method can also encompass a step ofadministering the first or additional therapeutic dose of acellularpro-tolerogenic preparation to the subject in need thereof. In someembodiments, the method can also encompass identifying if the subject isin need of increasing its Tregs to pro-inflammatory T cells ratio priorto the administration of the first or any therapeutic dose of theacellular pro-tolerogenic preparation. This can be done by actuallycharacterizing T cell subpopulations in the subject intended to betreated, measuring such ratio (and comparing it to ratios associatedwith age- and sex-matched healthy subjects) and/or identifying acondition which would require immune tolerance (e.g., the presence of anauto-immune condition for example).

In a second embodiment, the method allows to modulation of the ratio ofTreg to pro-inflammatory T cells in a subject having received a first(or multiple) therapeutic dose of an acellular pro-inflammatorypreparation. The method thus allows, in such subject, an increase in theratio of Treg to pro-inflammatory T cells which was previouslyintentionally decreased (by the administration of the acellularpro-inflammatory preparation). In order to achieve this increase in theratio of Treg to pro-inflammatory T cells, at least one therapeuticamount of at least one acellular pro-tolerogenic is administered.Further modulation of the ratio can be achieved by administeringsequentially and, optionally in an alternate fashion, additionaltherapeutic doses of the acellular pro-inflammatory preparations and theacellular pro-tolerogenic preparations. In some embodiments, in someembodiments, it may be advantageous to determine, prior to theadministration of a first or any acellular pro-tolerogenic preparation,if the subject is in need of increasing its Tregs to pro-inflammatory Tcells ratio. This can be done by actually measuring such ratio (andcomparing it to ratios associated with age- and sex-matched healthysubjects) and/or identifying a condition which would require immunetolerance (e.g., the presence of an auto-immune condition, a cell ortissue transplant, a risk of developing GVHD for example). The methodcan also encompass administering a first or additional therapeutic dosesof an acellular pro-inflammatory preparation to the subject. In someembodiments, it may be advantageous to determine, prior to theadministration of the first or any therapeutic dose of the acellularpro-inflammatory preparation, if the subject is in need of decreasingits Tregs to pro-inflammatory T cells ratio. This can be done byactually measuring such ratio (and comparing it to ratios associatedwith age- and sex-matched healthy subjects) and/or identifying acondition which would require immune stimulation (e.g., an infection,cancer progression or a vaccine for example).

(iv) Therapeutic Applications Aimed at Decreasing the Treg toPro-Inflammatory T Cell Ratio

In the present disclosure, the ratio can be decreased either by loweringthe level of regulatory T cells in the subject or increasing the levelof pro-inflammatory T cells in the subject. Alternatively, the ratio canbe decreased by lowering the level of regulatory T cells in the subjectand increasing the level of pro-inflammatory T cells in the subject.

When the Treg/pro-inflammatory T cells ratio is decreased in a subject,it is considered that a state of immune stimulation is induced orpresent in the subject. The induction of a state of immune stimulationin subjects experiencing an abnormally decreased immune state can betherapeutically beneficial for limiting the symptoms or pathologyassociated with the abnormally low immune reaction or an acquired stateof anergy. In some embodiments, it is not necessary to induce a state ofcomplete immune stimulation, a partial induction of immune stimulationcan be beneficial to prevent, treat and/or alleviate the symptoms of adisorder associated with a pro-tolerogenic state (such as, for example,a proliferation-associated disorder or an infection).

As shown herein, the administration of the acellular pro-inflammatorypreparations induces a state of immune stimulation in the treatedsubject. In some embodiments, the state of stimulation can persist longafter the administration of the acellular preparations (as shown below,at least 270 days in mice). Consequently, the methods and acellularpreparations described herein are useful for the treatment, preventionand/or alleviation of symptoms associated with conditionscaused/exacerbated by a low or inappropriate immune response.

A state of immune stimulation can be considered therapeuticallybeneficial in subjects experiencing a repressed immune response (anergyor tolerance), such as for example those observed upon the induction andmaintenance of a proliferation-associated disorder (such as cancer).Some of these conditions are associated with either a high level ofTregs and/or a low level of pro-inflammatory T cells (such as Th17and/or Th1) when compared to sex- and aged-matched healthy subjects.Because it is shown herein that the acellular-based preparations arebeneficial for decreasing the ratio Tregs/pro-inflammatory T cells, itis expected that administration of the acellular-based preparations toafflicted subjects will treat, prevent and/or alleviate symptomsassociated with the proliferation-associated disorder.

A state of immune stimulation can also be considered therapeuticallybeneficial in subjects at risk of developing an abnormally repressedimmune response, a state or anergy or a pro-tolerogenic state. Suchabnormally repressed immune responses can be observed in subjects beingafflicted by or susceptible to be afflicted by aproliferation-associated disorder such as cancer. In some embodiments,the acellular pro-inflammatory preparations are used for the treatment,prevention and/or alleviations of symptoms of non-blood cancer (e.g.solid cancers), such as, for example, carcinoma, melanoma, sarcoma,blastoma and germ-cell tumors. In this embodiment, the methods can beapplied to prevent or limit the onset or maintenance of a repressedimmune response. The acellular pro-tolerogenic preparations can beco-administered with the other therapeutics currently used to manage theproliferation-associated disorder.

The acellular-based preparation can be administered to any subjects inneed thereof, including humans and animals.

Such abnormally repressed immune responses can be also observed insubjects being infected, especially by a parasite or a virus. In theseconditions, the methods and acellular preparations can be applied toprevent or limit the onset or maintenance of a repressed immuneresponse. The acellular-based preparation can be co-administered withthe other therapeutics currently used to manage the infection.

In an embodiment, the state of abnormal repression of the immune systemis not caused by an infection of the immune cells themselves (e.g. EBVor HIV for example). However, in other embodiments, in instances wherean infection of the immune cells is afflicting the subject, it ispossible to use acellular pro-inflammatory preparations described totreat or alleviate the symptoms of the viral infection. For example, aleukocyte from the subject (preferably a cytotoxic T cell which isspecific to the infectious agent) can be co-cultured with the acellularpro-inflammatory preparations described herein. After the co-culture,the cultured leukocyte can be reintroduced in the infected subject totreat and/or alleviate the symptoms associated to the infection (a viralinfection, for example, an EBV or HIV infection). In another example,the acellular pro-inflammatory preparations described herein can beadministered to the infected individual to provide immune stimulation.

In the methods preparations described herein, it is contemplated thatthe acellular-based preparations be optionally administered with othertherapeutic agents known to be useful for the treatment, preventionand/or alleviation of symptoms of conditions associated to a conditioncaused/exacerbated by a low or inappropriate immune response, such as,for example, IL-2, IL-4, TNF-α and/or INF-γ.

(v) Therapeutic Applications Aimed at Increase in Treg toPro-Inflammatory T Cell Ratio

In the present disclosure, the ratio can be increased either byincreasing the level of regulatory T cells in the subject or decreasingthe level of pro-inflammatory T cells in the subject. Alternatively, theratio can be increased by increasing the level of regulatory T cells inthe subject and decreasing the level of pro-inflammatory T cells in thesubject.

When the Treg/pro-inflammatory T cells ratio is increased in a subject,it is considered that a state of immune tolerance is induced or presentin the subject. The induction of a state of immune tolerance in subjectsexperiencing an abnormally elevated immune state can be therapeuticallybeneficial for limiting the symptoms or pathology associated with apathological pro-inflammatory state (such as, for example, anauto-immune disease or an excessive immune response). In someembodiments, it is not necessary to induce a state of complete immunetolerance (e.g., anergy), a partial induction of immune tolerance can bebeneficial to prevent, treat and/or alleviate the symptoms of a disorderassociated with a pathological pro-inflammatory state.

Auto-immunity arises consequent to an animal/individual's immune systemrecognizing their own tissues as “non-self”. Autoimmunity is largely acell-mediated disease with T lymphocytes playing a central role in“self” recognition and are, in many cases, also the effector cells. TheNon-Obese Diabetic (NOD) mouse is an inbred strain that exhibits thespontaneous development of a variety of autoimmune diseases includinginsulin dependent diabetes. It is considered to be an exemplary mousemodel of autoimmunity in general. The murine autoimmune diabetesdevelops beginning around 10 to 15 weeks of age and has been extensivelyused to study the mechanisms underlying autoimmune-mediated diabetes,therapeutic interventions and the effect of viral enhancers on diseasepathogenesis. Diabetes develops in NOD mice as a result of insulitis, aleukocytic infiltrate of the pancreatic islets. This can be exacerbatedif mice are exposed to killed mycobacterium or other agents (Coxsackievirus for example). Multiple studies have established that thepathogenesis of diabetes in the NOD mouse is very similar to thatobserved in human type I diabetes (T1D) in that it is characterized bythe breakdown of multiple tolerance pathways and development of severeinsulitis of the islets prior to β-cell destruction. Moreover, T cells(including Th1, Th17 and Tregs) have been identified as key mediators ofthe autoimmune disease process though other cells (NK cells, B-cells, DCand macrophages) are also observed. Indeed, the NOD mouse model hastranslated into successful clinical human trials utilizing T-celltargeting therapies for treatment of many autoimmune diseases, includingT1D. The loss of function arising from pro-inflammatory allo-recognitionis exemplified by the destruction of the islets of Langerhans (insulinsecreting β cells) in the pancreas of the NOD mice leading to the onsetof Type 1 diabetes. In the context of type I diabetes, pro-tolerogenicallo-recognition is going to confer the protection and survival of theislets of Langerhans and the inhibition of diabetes in the treatedsubject.

A state of anergy or immune tolerance can be considered therapeuticallybeneficial in subjects experiencing (or at risk of experiencing) anabnormal immune response, such as for example an auto-immune disease.Individuals afflicted by auto-immune diseases have either low levels ofTregs and/or elevated levels of pro-inflammatory T cells (such as Th17and/or Th1) when compared to age- and sex-matched healthy individuals.Such auto-immune diseases include, but are not limited to, type Idiabetes, rheumatoid arthritis, multiple sclerosis, lupus, immunethrombocytopenia, experimental autoimmune encephalomyelitis, auto-immuneuveitis, psoriasis inflammatory bowel disease, scleroderma and Crohn'sdisease. Because it is shown herein that the acellular preparations arebeneficial for increasing the ratio Tregs/pro-inflammatory T cells, itis expected that administration of the acellular preparations toafflicted subjects will alleviate symptoms associated with theauto-immune disease and/or prevent disease severity.

A state of anergy or tolerance can also be considered therapeuticallybeneficial in subjects at risk of developing an abnormallyelevated/excessive immune response. Such abnormally elevated immuneresponse can be observed in subjects receiving a vaccine. For example,it has been shown that subjects receiving a respiratory syncytial virus(RSV) vaccine develop an excessive immune response. Because it is shownherein that the acellular preparations are beneficial for increasing theratio Tregs/pro-inflammatory T cells, it is expected that administrationof the acellular preparations to subject having received or intended toreceive a vaccine will alleviate symptoms associated with theadministration of the vaccine and/or prevent the development of anexcessive immune response. In such embodiment, the acellular preparationcan be administered (or formulated for administration) prior to thevaccine, simultaneously with the vaccine or after the administration ofthe vaccine. When used to prevent or limit excessive immune response toa vaccine, the acellular preparations can be manufactured from aconditioned medium. The conditioned medium can be obtained byco-culturing a first leukocyte, being allogeneic or xenogeneic to asecond leukocyte, which can be allogeneic, xenogeneic, autologous orsyngeneic to the subject to be vaccinated. The second leukocyte, muchlike the first leukocyte, can be allogeneic, xenogeneic, autologous orsyngeneic to the subject to be vaccinated. When used to prevent or limitan excessive immune response to a vaccine, the acellular preparationscan also be manufactured from a conditioned blood. The conditioned bloodcan be obtained by administered a first leukocyte, being allogeneic orxenogeneic to the test subject, which can be allogeneic, xenogeneic,autologous or syngeneic to the subject to be vaccinated.

Such abnormally elevated immune response can also be observed insubjects having received a transplant (cells or tissues). In theseinstances, the acellular preparations can be used to prevent or limitthe elevated/excessive immune response (e.g. graft destruction or graftrejection). In an embodiment, the acellular preparation can be contactedwith the cells/tissue to be transplanted prior to the transplantation(e.g. for example in a transplant medium or a preservation medium). Whenused to prevent or limit graft destruction or graft rejection, theacellular preparations can be manufactured from a conditioned medium.The conditioned medium can be obtained by co-culturing a firstleukocyte, being allogeneic or xenogeneic to a second leukocyte, whichcan be allogeneic, xenogeneic, autologous or syngeneic to the subject tobe treated. Alternatively, the first leukocyte is allogeneic,xenogeneic, autologous or syngeneic to the cells or tissue intended tobe grafted. The second leukocyte, much like the first leukocyte, can beallogeneic, xenogeneic, autologous or syngeneic to the subject to betreated. Alternatively, the second leukocyte is allogeneic, xenogeneic,autologous or syngeneic to the cells or tissue intended to be grafted.When used to prevent or limit graft destruction or graft rejection, theacellular preparations can also be manufactured from a conditionedblood. The conditioned blood can be obtained by administering a firstleukocyte, being allogeneic or xenogeneic to the test subject, which canbe allogeneic, xenogeneic, autologous or syngeneic to the subject to betreated. Alternatively, the first leukocyte is allogeneic, xenogeneic,autologous or syngeneic to the cells or tissue intended to be grafted.

Alternatively or optionally, the acellular preparations can also be usedto prevent or limit a graft-vs.-host disease (GVHD) in a subject havingreceived or intended to receive transplanted immune cells or stem cells.In an embodiment, the acellular preparations can be contacted (e.g.cultured) with the cells intended to be grafted prior to transfusion inthe subject (e.g. for example in a transplantation medium orpreservation medium) to induce a state of anergy or tolerance in thosecells. In another embodiment, the acellular preparations can beadministered to the subject prior to the transfusion of immune/stemcells to induce a state of anergy or tolerance to prevent or limit GVHD.In still another embodiment, the acellular preparations can beadministered simultaneously with the transfused immune/stem cells toprevent or limit GVHD. In yet another embodiment, the acellularpreparations can be administered to a subject having been transfusedwith immune cells or stem cells either to alleviate the symptomsassociated to GVHD (when the subject experiences such symptoms) or toprevent GVHD (when the subject is at risk of experiencing suchsymptoms).

For the treatment of GVHD, the conditioned medium can be obtained byco-culturing two allogeneic/xenogeneic leukocyte populations. In anembodiment, the first leukocyte population can be allogeneic,xenogeneic, syngeneic to or derived from the donor (of the immune orstem cells). In another embodiment, the first leukocyte population canbe allogeneic, xenogeneic, syngeneic to or derived from the recipient(intended to receive the immune or stem cells). In still anotherembodiment, the second leukocyte population can be allogeneic,xenogeneic, syngeneic to or derived from the donor. In yet anotherembodiment, the second leukocyte population can be allogeneic,xenogeneic, syngeneic to or derived from the recipient. For thetreatment of GVHD, the conditioned blood can be obtained byadministering a first leukocyte allogeneic or xenogeneic to the testsubject (e.g. and in an embodiment to the donor). In an embodiment, thefirst leukocyte population can be allogeneic, xenogeneic, syngeneic toor derived from the donor. In another embodiment, the first leukocytepopulation can be allogeneic, xenogeneic, syngeneic to or derived fromthe recipient. The acellular preparation can be administered (orformulated for administration) prior to the transplant, simultaneouslywith the transplant or after the transplant.

In the methods described herein, it is contemplated that the acellularpro-tolerogenic preparations be optionally administered with othertherapeutic agents known to be useful for the treatment, preventionand/or alleviation of symptoms of conditions associated to anexcessive/abnormal immune response, such as, for example, cortisone,IL-10, IL-11 and/or IL-12.

The present invention will be more readily understood by referring tothe following examples which are given to illustrate the inventionrather than to limit its scope.

Example I—Material and Methods Human PBMC and Dendritic CellPreparation.

Human whole blood was collected in heparinized vacutainer bloodcollection tubes (BD, Franklin Lakes, N.J.) from healthy volunteerdonors following informed consent. PBMC were isolated from diluted wholeblood using FicollePaque PREMIUM™ (GE Healthcare Bio-Sciences Corp,Piscataway, N.J.) as per the product instructions. The PBMC layer waswashed twice with 1× Hank's Balanced Salt Solution (HBSS; without CaCl₂)and MgSO₄; Invitrogen by Life Technologies, Carlsbad, Calif.) andresuspended in the appropriate media as needed for mixed lymphocytereactions and flow cytometric analysis of Treg and Th17 phenotypes.Dendritic cells (DC) were prepared from isolated PBMC as described byO'Neill and Bhardwaj (O'Neill et al., 2005). Briefly, freshly isolatedPBMC were overlaid on Petri dishes for 3 h in AIM V serum free culturemedium (Invitrogen, Carlsbad, Calif.). Non-adherent cells were gentlywashed off the plate. The adherent cells (monocyte rich cells) weretreated with IL-4 and GM-CSF (50 and 100 ng/mL respectively; R&DSystems, Minneapolis, Minn.) in AIM V medium. Cells were again treatedwith IL-4 and GM-CSF on days 2 and 5. On day 6, cells were centrifugedand resuspended in fresh media supplemented with DC maturation factors(TNF-α, IL-1β, IL-6; R&D Systems, Minneapolis, Minn.) and prostaglandinE2 (Sigma Aldrich, St. Louis, Mo.). The mature DC-like cells wereharvested on day 7 and CD80, CD83, CD86 and HLA-DR expressions weredetermined to confirm DC maturation via flow cytometry (FACSCalibur™Flow Cytometer, BD Biosciences, San Jose, Calif.).

Murine Splenocyte and Tissue Harvesting.

All murine studies were done in accordance with the Canadian Council ofAnimal Care and the University of British Columbia Animal Care Committeeguidelines and were conducted within the Centre for Disease Modeling atthe University of British Columbia. Murine donor cells used for the invivo donation and in vitro studies were euthanized by CO₂. Threeallogeneic strains of mice were utilized for syngeneic and allogeneic invitro and in vivo challenge: Balb/c, H-2^(d); C57Bl/6, H-2^(b); and C3H,H-2^(k). Murine spleens, brachial lymph nodes, and peripheral blood werecollected at the indicated days. Mouse spleens and brachial lymph nodeswere dissected and placed into cold phosphate buffered saline (PBS; 1.9mM NaH₂PO₄, 8.1 mM Na₂HPO₄, and 154 mM NaCl, pH 7.3) containing 0.2%bovine serum albumin (BSA; Sigma Aldrich, St. Louis, Mo.) and kept onice until ready to process. Whole blood was collected in heparinizedtubes via cardiac puncture. Murine donor splenocytes were prepared fromfreshly harvested syngeneic or allogeneic spleens via homogenizationinto a cell suspension in PBS (0.2% BSA) using the frosted end of twomicroscope slides. The resultant cell suspension was spun down at 500×g.The splenocyte pellet was resuspended in 1 mL of 1×BD Pharm LYSE™ lysingbuffer (BD Biosciences, San Diego, Calif.) and incubated for 1 min atroom temperature. Lymph node cells were harvested via tissuehomogenization as described above, washed twice and resuspended in PBS(0.2% BSA) for flow cytometric analysis of Th17, Treg and murinehaplotype. Recipient peripheral blood lymphocytes were prepared vialysis of the red cells (BD Pharm Lyse lysing buffer; BD Biosciences, SanDiego, Calif.) at 1× concentration, followed by washing (1×) andresuspension in PBS (0.2% BSA) for flow analysis of Th17, Treg andmurine haplotype.

mPEG Modification (PEGylation) of PBMCs and Splenocytes.

Human PBMC and murine splenocytes were derivatized usingmethoxypoly(-ethylene glycol) succinimidyl valerate (mPEG-SVA; LaysanBio Inc. Arab, Ala.) with a molecular weight of 5 or 20 kDa aspreviously described (Scott et al., 1997; Murad et al, 1999; Chen etal., 2003; Chen et al., 2006). Grafting concentrations ranged from 0 to5.0 mM per 4×10⁶ cells/mL. Cells were incubated with the activated mPEGfor 60 min at room temperature in isotonic alkaline phosphate buffer (50mM K₂HPO₄ and 105 mM NaCl; pH 8.0), then washed twice with 25 mMHEPES/RPMI 1640 containing 0.01% human albumin. Human PBMC wereresuspended in AIM V media at a final cell density of 2.0×10⁶ cells/mLfor use in the MLR. Murine splenocytes used for in vivo studies wereresuspended in sterile saline at a final cell density of 2.0×10⁸cells/ml for i.v. injection. To determine if the simple presence of themPEG polymer itself altered the immune response either in vitro and invivo, additional studies were done with unactivated polymer incapable ofcovalent grafting to the cell surface. For these studies, allogeneichuman (in vitro studies) or syngeneic and allogeneic murine splenocytes(in vivo studies) were treated with non-covalently bound mPEG (solublemPEG) under the same reaction conditions described for the covalentgrafting studies. For clarity, “soluble mPEG” refers to cells treatedwith non-covalently grafted polymer while “mPEG-modified” refers totreatment with activated polymer resulting in the covalent grafting ofthe mPEG to the cell membrane.

In Vitro and In Vivo Cell Proliferation.

Cell proliferation (both in vitro and in vivo) was assessed via flowcytometry using the CELLTRACE™ CFSE (Carboxyfluorescein diacetate,succinimidyl ester) Cell Proliferation Kit (Invitrogen by LifeTechnologies e Molecular probes, Carlsbad, Calif.). Human and murinecells labeling was done according to the product insert at a finalconcentration of 2.5 mM CFSE per 2×10⁶ cells total. Donor and recipientcell proliferation was differentially determined by haplotype analysis.In some experiments, cell proliferation was measured by ³H-thymidineincorporation. In these experiments, donor splenocytes (5.12×10⁶ cellsper well) were co-incubated in triplicate in 96-well plates at 37° C.,5% CO₂ for 3 days. On day 3, all wells were pulsed with ³H-thymidine andincubated for 24 h at 37° C., 5% CO₂. Cellular DNA was collected onfilter mats using a Skatron cell harvester (Suffolk, U.K.) and cellularproliferation was measured by ³H-thymidine incorporation.

Mixed Lymphocyte Reaction (MLR)—Control and Conditioned Media.

The immunomodulatory effects of the various preparations were assayedusing a MLR (Murad et al, 1999; Chen et al., 2003; Chen et al., 2006;Wang et al., 2011). For the human MLRs, PBMC from two MHC-disparatehuman donors were labeled with CFSE. For mice MLR, splenocytes from twoH-2-disparate mice (Balb/c and C57Bl/6) were labeled with CFSE. Each MLRreaction well contained a total of 1×10⁶ cells (single donor for restingor mitogen stimulation or equal numbers for disparate donors for MLR).Cells were plated in multiwell flat-bottom 24-well tissue culture plates(BD Biosciences, Discovery Labware, Bedford, Mass.). PBMC proliferation,cytokine secretion, as well as Treg and Th17 phenotyping was done. Forflow cytometric analysis, the harvested cells were resuspended in PBS(0.1% BSA).

Immunophenotyping by Flow Cytometry.

The T lymphocytes populations (double positive for CD3⁺ and CD4⁺) inboth the in vitro and in vivo studies were measured by flow cytometryusing fluorescently labeled CD3 and CD4 monoclonal antibodies (BDPharmingen, San Diego, Calif.). Human and mouse Regulatory T lymphocytes(Treg) were CD3⁺/CD4⁺ and FoxP3⁺ (transcription factor) whileinflammatory Th17 lymphocytes cells were CD3⁺/CD4⁺ and IL-17⁺ (cytokine)as measured per the BD Treg/Th17 Phenotyping Kit (BD Pharmingen, SanDiego, Calif.). Additional cell surface markers were also used tocharacterize the cells or subsets of cells obtained: anti-CD69 (cloneH1.2F3, BD Biosciences) and anti-CD25 (BD Biosciences). After thestaining, the cells (1×10⁶ cells total) were washed and resuspended inPBS (0.1% BSA) prior to flow acquisition. Isotype controls were alsoused to determine background fluorescence. All samples were acquiredusing the FACSCalibur™ flow cytometer (BD Biosciences, San Jose, Calif.)and CellQuest Pro™ software for both acquisition and analysis.

Conditioned Plasma.

Mouse were either untreated (naïve) or treated with saline, non-polymermodified allogeneic splenocytes or PEGylated allogeneic splenocytes(obtained by the procedures explained above). After five days, acell-free conditioned plasma was obtained (from mouse blood using themirVana™ PARIS™ kit from Ambion by Life Technologies) and transfused toanother naïve mouse.

Plasma Fractionation.

The plasma fractionation was performed using centrifugal filtermolecular cutoff devices. Millipore's Amicon® Ultra-0.5 centrifugalfilter devices were used (Amicon Ultra 3k, 10K, 30K, 50K, and 100Kdevices).

miRNA Extraction.

The miRNA was extracted from samples (conditioned medium or plasma)using mirVana™ PARIS™ kit from Ambion® by Life Technologies according tothe manufacturer's instructions. Briefly, the sample is mixed with the2× denaturing solution provided and subjected to acid-phenol:chloroformextraction. To isolate RNA that is highly enriched for small RNAspecies, 100% ethanol was added to bring the samples to 25% ethanol.When this lysate/ethanol mixture was passed through a glass-fiberfilter, large RNAs are immobilized, and the small RNA species arecollected in the filtrate. The ethanol concentration of the filtrate wasthen increased to 55%, and it was passed through a second glass-fiberfilter where the small RNAs become immobilized. This RNA is washed a fewtimes, and eluted in a low ionic strength solution. Using this approach,an RNA fraction highly enriched in RNA species<200 nt can be obtained.Note that the large RNA species (>200 nt) can be recovered from thefirst filter if necessary.

TA Preparations.

The murine miRNA preparations (e.g. TA1 preparations) used wereextracted from the conditioned plasma obtained 5 days after mice havereceived mPEG allogeneic splenocytes. Extraction can occur at timepoints other than 5 days (e.g., 24 hours post administration) and yieldsimilar results (data not shown). Five days was chosen as Treg levelsachieved maximal levels at this point in the mice. The human miRNApreparations (e.g. TA2 preparations) used were extracted from theconditioned medium of an mPEG-MLR harvested 72 hours following theinitiation of the mPEG-MLR. However, miRNA harvested from human PBMCmPEG-MLR at 24 hours also yields the desired immunomodulatory effects(data not shown). To calibrate, miRNA concentration can be quantitatedvia a Qubit® 2.0 Fluorometer (LifeTechnologies) and selected fluorescentdyes which emit a signal only when bound to specific target (i.e.,miRNA) molecules.

IA Preparations.

The murine miRNA preparations (e.g. IA1 preparations) used wereextracted from the conditioned plasma obtained 5 days after mice havereceived non-polymer modified allogeneic splenocytes. Extraction canoccur at time points other than 5 days (e.g., 24 hours postadministration) and yield similar results (data not shown). Five dayswas chosen as Th17 levels achieved maximal levels and Treg cells hadreach their minimal level at this point in the mice. The human miRNApreparations (e.g. IA2 preparations) used were extracted from theconditioned medium of an mPEG-MLR harvested 72 hours following theinitiation of the mPEG-MLR. However, miRNA harvested from human PBMCmPEG-MLR at 24 hours also yields the desired immunomodulatory effects(data not shown). To calibrate, miRNA concentration can be quantitatedvia a Qubit® 2.0 Fluorometer (LifeTechnologies) and selected fluorescentdyes which emit a signal only when bound to specific target (i.e.,miRNA) molecules.

miRNA Characterization.

The miRNA of the conditioned medium were characterized by qPCR using themiScript miRNA™ PCR Array Human Immunopathology (Qiagen) for humanconditioned medium and the Mouse Immunopathology miRNA PCR Array™(Qiagen) for mouse conditioned plasma/media.

RNase Treatment.

Murine plasma was pooled and for each individual mouse. For each 500 μLof murine plasma (or the <10 kDa plasma fraction), 50 ng RNase (RNase A,20 mg/mL stock, Life Technologies (In Vitrogen)) was added. Then sampleswere incubated for 10 minutes at 37° C. to degrade the nucleic acids.The control plasma (or <10 kDa fraction) without RNAase A treatment wasincubated at 37° C. for 10 min. The RNase treated plasma (100 μl permouse) was injected (i.v.) into mice (n=5). RNase A alone (10 ng/mouse)was used for the control mice to insure that the RNase A was not toxicand this trace amount of RNase did not have an in vivo immunomodulatoryeffects.

Phosphorylation of Phosphokinases.

Analyzing the phosphorylation state of kinases and their proteinsubstrates allows for the characterization of the effects of conditionedplasma or media on how cells respond to allogeneic stimuli. The humanphospho-kinase array (R&D Systems Inc) is a rapid, sensitive tool tosimultaneously detect the relative levels of phosphorylation of 43kinase phosphorylation sites and 2 related total proteins. Each captureantibody was carefully selected using cell lysates prepared from celllines known to express the target protein. Capture and controlantibodies are spotted in duplicate on nitrocellulose membranes. Celllysates are diluted and incubated overnight with the humanphospho-kinase array. The array is washed to remove unbound proteinsfollowed by incubation with a cocktail of biotinylated detectionantibodies. Streptavidin-HRP and chemiluminescent detection reagents areapplied and a signal is produced at each capture spot corresponding tothe amount of phosphorylated protein bound.

Statistical Analysis.

Data analysis for flow analysis was conducted using SPSS™ (v12)statistical software (Statistical Products and Services Solutions,Chicago, Ill., USA). For significance, a minimum p value of <0.05 wasused. For comparison of three or more means, a one-way analysis ofvariance (ANOVA) was performed. When significant differences were found,a post-hoc Tukey test was used for pair-wise comparison of means. Whenonly two means were compared, student-t tests were performed.

In Vivo Murine Studies.

Three genetically different strains: Balb/c, H-2^(d); C57Bl/6, H-2^(b);and C3H, H-2^(k) (Chen et al., 2003; Chen et al., 2006). All mice(donors and recipients) were 9-11 weeks old. Donor splenocytes wereprepared and CSFE labeled as described. control and mPEG-grafted (1 mM,20 kDa SVAmPEG) syngeneic or allogeneic cells (20×10⁶ splenocytes) weretransfused intravenously (i.v.) via the tail vein into recipientanimals. BALB/c and C57BL/6 mice injected with sterile saline served ascontrol animals. Animals were euthanized by CO₂ at predeterminedintervals at which time blood, brachial lymph nodes and spleen werecollected and processed for Th17/Treg phenotyping analysis andsplenocyte proliferation studies by flow cytometry. Donor cellengraftment and proliferation were assessed via flow cytometry usingmurine haplotype (H-2K^(b) vs. H-2K^(d)) analysis. To determine thepersistence of the immunomodulation, mice were re-challenged (2°challenge) 30 days after the initial transfer of allogeneic or mPEGallogeneic splenocytes with unmodified allogeneic cells. At 5 days post2° challenge, Treg and Th17 phenotyping of murine splenocytes isolatedfrom the spleen, lymph node and peripheral blood was again assessed viaflow cytometry.

Statistical Analysis.

Data analysis was conducted using SPSS™ (v12) statistical software(Statistical Products and Services Solutions, Chicago, Ill., USA). Forsignificance, a minimum p value of <0.05 was used. For comparison ofthree or more means, a one-way analysis of variance (ANOVA) wasperformed. When significant differences were found, a post-hoc Tukeytest was used for pair-wise comparison of means. When only two meanswere compared, student-t tests were performed.

Example II—Characterization of Conditioned Plasma Obtained fromAdministering Polymer-Grafted and Non-Polymer-Grafted Lymphocytes

The material and methods used in this example are provided in Example I.

In a first series of experiment, several types of conditioned plasmawere obtained. Briefly, the conditioned plasma was first obtained fromBalb/c mice having received saline, 20×10⁶ allogeneic (C57BL/6)PEGylated splenocytes (using 1 mM 20 kDa PEG) or 20×10⁶ allogeneic(C57BL/6) unmodified splenocytes. The conditioned plasma was either leftuntreated (e.g. complete) or fractionated in function of the size of itscomponents (>100 kDa, between 30 and 100 kDa, between 10 and 30 kDa or<10 kDa). The saline of the treated conditioned plasma was thentransfused to naïve C57BL/6 mice. Five days after the administration,the animals were sacrificed, the spleen was obtained and the cells theycontained was characterized. As shown on FIG. 1A, the <10 kDa fractionof the conditioned plasma from mouse having received unmodifiedallogeneic splenocytes retained the ability, when compared to completeunfractionated conditioned plasma, to increase Th17 levels in vivo. Asshown on FIG. 1B, the <10 kDa fraction of the conditioned plasma frommouse having received unmodified allogeneic splenocytes retained theability, when compared to complete unfractionated conditioned plasma, todecrease Treg levels in vivo. The immunomodulatory effect of conditionedmurine plasma seems to mostly reside in the lower molecular weightfraction (<10 kDa). This low molecular weight fraction does not includethe majority of cytokines (usually encompassed in the 100-30 and the30-10 kDa fractions) typically thought to mediate immunomodulation ofTregs and pro-inflammatory leukocytes. However, the <10 kDa fraction issuspected to contain, among its components, microRNAs (miRNAs). Todetermine if the miRNAs in the conditioned plasma mediated theimmunomodulatory effects observed with the conditioned plasma, mice wereinjected with untreated conditioned plasma or conditioned plasma thathad been pre-treated with RNase A, an enzyme that degrades/destroysribonucleic acids such as miRNAs. As noted in FIG. 10, treatment withRNase A greatly reduced the immunomodulatory activity of the conditionedplasma, thereby confirming the ribonucleic acid nature of thesize-fractionated conditioned plasma.

To determine the effects of acellular components obtained fromadministering polymer-grafted and non-polymer grafted (e.g., unmodified)allogeneic lymphocytes on the immune response, a second series of invivo experiments was conducted. The conditioned plasma was firstobtained from Balb/c mice having received saline, 20×10⁶ allogeneic(C57BL/6) PEGylated splenocytes (using 1 mM 20 kDa PEG) or 20×10⁶allogeneic (C57BL/6) unmodified splenocytes. Saline or one of theconditioned plasma obtained was administered to Balb/c mice either once(at day 0) or thrice (at days 0, 2 and 4). Five days after the lastadministration, the animals were sacrificed, the spleen and the brachiallymph node were obtained and the cells they contained was characterized.More specifically, the percentage of Foxp3⁺, CD69⁺ or CD25⁺ cells (withrespect to the total number of CD4⁺ recuperated) was determined. Asshown in FIG. 2A, the number of CD25⁺ cells was reduced in animalshaving received the conditioned plasma obtained from administeringunmodified allogeneic splenocytes (Plasma (Allo) bars on FIG. 2A). Onthe other hand, the number of CD25⁺ cells was elevated in animals havingreceived the conditioned plasma obtained from administering PEGylatedallogeneic splenocytes (Plasma (mPEG-Allo) bars on FIG. 2A). Thepopulation of CD25⁺ cells includes Foxp3⁺ as well as Foxp3⁻ Treg cells.These findings suggest that the conditioned plasma obtained fromadministering unmodified allogeneic splenocyte reduces Treg cell levelsand induces a pro-inflammatory immune reaction. These findings alsosuggests that the conditioned plasma obtained from administeringpolymer-modified allogeneic splenocyte increases Treg cell levels andinduces an anti-inflammatory immune reaction.

As shown in FIG. 2B, the number of CD69⁺CD25⁻ cells (e.g., non-Foxp3Tregs) was reduced in animals having received the conditioned plasmaobtained from administering unmodified allogeneic splenocytes (Plasma(Allo) bars on FIG. 2B) and increased in animals having received theconditioned plasma obtained from administering polymer-modifiedallogeneic splenocytes (Plasma (mPEG-Allo) bars on FIG. 2B). Non-Foxp3Tregs are known to be elevated in tumor-bearing mouse models arebelieved to limit/prevent the tumor regression in these animals. Thesefindings suggest that the conditioned plasma obtained from administeringunmodified allogeneic splenocytes decreases the level of such Tregsubset and may be beneficial in facilitating tumor regression intumor-bearing animals. These findings also suggest that the conditionedplasma obtained from administering polymer-modified allogeneicsplenocytes increases the level of such Treg subset and may bebeneficial in establishing an immune tolerance.

FIG. 2C compiles the data presented in FIGS. 2A and 2B and shows thevarious Treg subsets (Foxp3+, CD25+ and CD69+ cells as a percentage ofthe total CD4+ cells) in the spleen and in the brachial lymph nodes ofthe treated animals.

The size-fractionation conditioned plasma was administered to mice andits effects on the intracellular cytokine expression of CD4⁺ cells wereexamined. As shown on FIG. 3A, the <10 kDa fraction and some of the <3kDa fraction of the conditioned plasma from mouse having receivedunmodified allogeneic splenocytes do not modulate IL-10 intracellularexpression in CD4⁺ cells in vivo, whereas the <10 kDa fraction and someof the <3 kDa fraction of the conditioned plasma from mouse havingreceived polymer-modified allogeneic splenocytes do modulate IL-10intracellular expression in CD4⁺ cells in vivo. In contrast, the <10 kDafraction and the <3 kDa fraction of the conditioned plasma from mousehaving received unmodified allogeneic splenocytes increased IL-2, TNF-α,IFN-γ and IL-4 intracellular expression in CD4⁺ cells in vivo, whereasthe <10 kDa fraction and the <3 kDa fraction of the conditioned plasmafrom mouse having received unmodified allogeneic splenocytes increasedIL-2, TNF-α, IFN-γ and IL-4 intracellular expression in CD4⁺ cells invivo (FIGS. 3B to 3E). The <10 kDa (and some of the >3 kDa) fraction ofthe conditioned plasma derived from unmodified allogeneic splenocytes,when compared to the corresponding fractions of the conditioned plasmaderived from mPEG allogeneic splenocytes, increased the expression ofpro-inflammatory cytokines, such as IL-2, TNF-α, IFN-γ or IL-4. However,pro-tolerogenic cytokines in animals having received conditioned plasmaderived from unmodified allogeneic splenocytes remained at levels seenin naïve animals.

Example III—Characterization of miRNA Preparations Obtained withPolymer-Grafted or Non-Polymer-Grafted Lymphocytes

The material and methods used in this example are provided in Example I.

The conditioned plasma or the miRNA preparation (100 μL) obtained fromthe conditioned plasma (of mice having received saline, unmodifiedallogeneic splenocytes or polymer-modified allogeneic splenocytes) wereadministered intravenously to 7-8 week-old mice thrice (at days 0, 2 and4). Cohorts (n=4) of mice were sacrificed at days 30, 60, 120, 180 and270. Spleens were removed and CD4⁺ cells were stained for intracellularexpression of IL-2, IL-4, IL-10, INF-γ and TNF-α. Splenic Treg and Th17populations were also measured. As shown on FIGS. 4A-C, theadministration of the conditioned plasma or the derived miRNApreparation (i.e., IA1 preparations) from mouse having receivedunmodified allogeneic splenocytes caused an increase in the expressionof intracellular IL-2 and INF-γ in CD4⁺ cells. On the other hand, theadministration of the conditioned plasma or the derived miRNApreparation from mouse having received mPEG-modified allogeneicsplenocytes (i.e., TA1 preparation) caused an increase in the expressionof intracellular IL-10 in CD4⁺ cells. These modulations in expressionwere observed until at least 270 days after the administration of theconditioned medium or the miRNA preparation. This data suggests thatmiRNA was an active component mediating the immunological changes, RNasetreatment of the conditioned plasma or of the miRNA preparation prior toadministration to animals either diminished (plasma) or abolished(miRNA) the immunomodulatory effects. While conditioned plasma retainedsome immunomodulatory effect, it is believed that it was due to residualcytokines and/or plasma-mediated inactivation of the RNAase A enzyme.

As also shown on FIG. 4D, the administration of the conditioned plasmaor the derived miRNA preparation from mouse having received unmodifiedallogeneic splenocytes (i.e., IA1 preparations) caused a decrease in thepercentage of Treg (Foxp3⁺) cells in function of the total CD4⁺ cells,whereas the administration of the conditioned plasma or the derivedmiRNA preparation from mouse having received polymer-modified allogeneicsplenocytes (i.e., TA1 preparations) caused an increase in thepercentage of Treg (Foxp3⁺) cells in function of the total CD4⁺ cells.In addition, the administration of the conditioned plasma or the derivedmiRNA preparation (i.e., IA1 preparations) from mouse having receivedunmodified allogeneic splenocytes caused an increase in the percentageof Th17 (IL-17⁺) cells in function of the total CD4⁺ cells, whereas theadministration of the conditioned plasma or the derived miRNApreparation (i.e., TA1 preparations) from mouse having receivedpolymer-modified allogeneic splenocytes caused a decrease in thepercentage of Th17 (IL-17⁺) cells in function of the total CD4⁺ cells(FIG. 4E). These modulations in CD4⁺ cells types were observed at least270 days after the administration of the conditioned medium or the miRNApreparations and were diminished (plasma) or abolished (miRNA) with apreliminary RNase treatment. Acellular preparations prepared from miceinjected with either allogeneic leukocytes exerted potent andlong-lasting effects in naive recipient mice. In aggregate, unmodifiedallogeneic-derived preparations (plasma or miRNA) yielded apro-inflammatory state while mPEG-allogeneic-derived preparations(plasma or miRNA) yielded a immunoquiescent state.

Murine (IA1 preparations) miRNA preparations exert a direct effect oncell signaling. Murine IA1 or TA1 preparations have been incubated withJurkat cells (1×10⁶ cells/ml treated with 50 μl of IA1 or TA1/ml) andthe level of phosphorylation of some of the phosphokinase has beenmeasured after 30 minutes of incubation. As shown on FIG. 5, IA1preparations favored the phosphorylation of the HSP60 and WNK1 kinases.As also shown on FIG. 5, TA1 preparations favored the phosphorylation ofthe Akt and PRAS40 while favoring the dephosphorylation of HSP60.

Murine IA1 preparations were also introduced (at time 0) into a humanPBMC MLR assay in order to determine their effect on humanallo-recognition. As indicated on FIG. 6, the presence of the murine IA1preparations resulted in a dose-dependent increase in the percentage inleukocyte proliferation (at both 10 and 14 days) which is indicative oftheir pro-inflammatory effects. This data also indicates that the IA1preparations show significant evolutionary conservations (both sequencespecific and similarity) since the murine IA1 preparations are highlyeffective in a xenogeneic system (e.g. human MLR).

Murine TA1 preparations were also introduced (at time 0) into a humanPBMC MLR assay in order to determine their effect on humanallo-recognition. As indicated on FIG. 6, the presence of the murine TA1preparations resulted in a dose-dependent decrease in the percentage inleukocyte proliferation (at both 10 and 14 days) which is indicative oftheir pro-tolerogenic effects. This data also indicates that the TA1preparations show significant evolutionary conservations (both sequencespecific and similarity) since the murine TA1 are highly effective in axenogeneic system (e.g. human MLR).

Murine IA1 or TA1 preparations (100 μL) were administered once (at day0) or thrice (at days 0, 2 and 4). Five days after the lastadministration, the mice were sacrificed, their spleen and brachiallymph nodes were obtained and the cells they contained werecharacterized. As described in PCT/CA2013/050963 filed on Dec. 13, 2013,IA1 preparations, unlike TA1 preparations, increased the percentage ofNK cells (as measured by flow cytometry) with respect to the totalnumber of CD4⁺ cells (data not shown). Similar results were obtained inthe spleen and the lymph nodes (data not shown).

While the murine TA1 preparation proved effective both in vitro and invivo in experimental models involving immunologically normal cells andanimals, to test the effectiveness of the TA1 preparation, a model ofautoimmune disease, NOD mice, were used. As described inPCT/CA2013/050545 filed on Jul. 12, 2013 and published underWO2014/008610 on Jan. 16, 2014, significant changes in the levels ofTh17 and Treg lymphocytes are noted in the spleen, brachial lymph nodeand pancreatic lymph nodes upon conversion of NOD mice from non-diabeticto diabetic state (data not shown). These changes are characterized bydramatically increased Th17 (top numbers in each panels) andsignificantly decreased Treg (lower numbers in each panels) lymphocytes.Murine TA1 preparations were administered intravenously once to NODmice. The administration of TA1 caused a shift in immune modulation atday 5 post treatment towards immune tolerance by decreasing thecirculating blood levels of pro-inflammatory Th17 cells (data notshown). The administration of the murine TA1 preparations to NOD miceyielded significant protection against progression to diabetes (data notshown). The administration of the murine TA1 preparations to NOD micecaused a systemic and/or local increase in pro-tolerogenic leukocytes(data not shown). The administration of the murine TA1 preparations toNOD mice also caused a decrease in pro-inflammatory Th17 cells and Th1cells as well as the decrease in the percentage of INF-γ⁺ cells, IL-2⁺cells, TNF-α⁺ cells and IL-12⁺ cells (data not shown). This data suggestthat the TA1 preparations prevented Th17/Th1 upregulation in the treatedmice, and ultimately prevented islet cell destruction. Interestingly,the administration of TA1s caused a significant increase in the level ofNK cells in the pancreas, but not in other tissues (data not shown). Itis believed that the differentially induced NK cells in the pancreasdestroys autoreactive (i.e. inflammatory) cells providing an additionalimmunomodulatory mechanism resulting in decreased diabetes. Further, ithas been shown are that the administration of TA1s increased B10⁺ (Bregulatory) cells and tolerogenic DC cell levels while decreasing APCassociated with inflammation (data not shown) further confirming thepro-tolerogenic effects of TA1s.

It was previously determined, for example in PCT/CA2013/050546 filed onJul. 12, 2013 and published as WO2014/008611 on Jan. 16, 2014, that ashift to a pro-inflammatory state caused by the administration ofnon-polymer-modified cellular (lymphocyte) preparations was long livedas well as systemic and that animals did not revert back to theirinitial immunological state upon the administration of apolymer-modified cellular (lymphocyte) preparation. It was alsopreviously determined, for example in PCT/CA2013/050546 filed on Jul.12, 2013 and published as WO2014/008611 on Jan. 16, 2014, that a shiftto a pro-tolerogenic state caused by the administration ofpolymer-modified cellular (lymphocyte) preparations was long lived aswell as systemic and that animals did not revert back to their initialimmunological state upon the administration of a unmodified cellular(lymphocyte) preparation. As such, it was thus determined if theacellular preparations described herein also induce a non-reversiblelong lived and systemic immune modulation.

In order to do so, the animals were divided into two groups: thosereceiving a single type of preparation and those receiving the twodifferent types of preparations (sequentially administered). Saline, TA1preparations (100 μL) or IA1 preparations (100 μL) were intravenouslyadministered to naïve mice at days 0, 2 and 4. Animals receiving only asingle type of preparations were then sacrificed at day 40. Animalsreceiving a second type of preparation were administered with IA1preparations (how much) or TA1 preparations (how much) at days 9, 11 and13 and were then sacrificed at day 40. Each treatment group contained atleast 5 animals. The percentage of Treg (FIGS. 7A and 7B) and Th17(FIGS. 7C and 7D) cells in the spleen and the brachial lymph nodes werethen determined for the mice of the different treatment groups. As shownin FIGS. 7A to 7D, the administration of TA1 preparations (alone) causedan increase in Treg levels and a decrease in Th17 levels whereas, theadministration of IA1 preparations (alone) caused a decrease in Treglevels and an increase in Th17 levels. It was surprisingly found thatwhen animals previously administered TA1 preparations (which caused ashift towards a pro-tolerogenic state) were subsequently challenged withIA1 preparations, their Treg levels decreased and their Th17 ratioincreased. On the other hand, when animals previously administered IA1preparations (which caused a shift towards a pro-inflammatory state)were subsequently challenged with TA1 preparations, their Treg levelsincreased and their Th17 levels decreased. These results are surprisingbecause, it was previously shown that a pro-tolerogenic state induced bythe administration of polymer-modified allogeneic cellular preparationscould not be shifted towards a more pro-inflammatory state upon theadministration of a non-modified allogeneic cellular preparations(WO2014/008611). However, as shown herein, the secondary administrationof IA1 was able to modulate the animals' immune response towards a morepro-inflammatory state once a pro-tolerogenic state has been induced.The results are also surprising because, it was previously shown that apro-inflammatory state induced by the administration of non-modifiedallogeneic cellular preparations could not be shifted towards a morepro-tolerogenic state upon the administration of polymer-modifiedallogeneic preparations (WO2014/008611). However, as shown herein, thesecondary administration of TA1 preparations was able to modulate theanimals' immune response towards a more pro-tolerogenic state once apro-inflammatory state has been induced.

Example IV—miRNA Characterization of Acellular ProtolerogenicPreparations

Some of the material and methods referred to in this example areprovided in Example I.

In order to characterize the constituents of the miRNA preparations, themiRNA of conditioned medium collected at 72 hours from resting humanPBMC, a human control MLR (using two HLA disparate PBMC populations),and a mPEG MLR (using the same two allogeneic PBMC populations whereinone population is modified with a polymer, e.g. mPEG) and compared viaqPCR analysis. The miRNA preparations obtained from the human controlMLR is referred to as IA2. The miRNA preparations obtained from thehuman mPEG MLR is referred to TA2. The combined average of the restingDonor A and resting Donor B (i.e., resting AB) were used, unlessotherwise noted, for baseline in all analyses.

As shown in FIG. 8A, when the IA2 miRNA population is compared to themiRNA population of the supernatant of resting cells, using a volcanoplot analysis, at least five different miRNAs are differentiallyexpressed (e.g. increased) by statistical significance (p<0.01 formiR-9-5p, miR-155-5p, miR-206, miR-147a and p<0.05 for miR-214-3p) andat least one miRNA is modulated by at least a log₂ (e.g. miR-302a-3p).

In contrast, as shown in FIG. 8B, when the TA2 miRNA population iscompared, using a volcano plot analysis, with the miRNA population ofthe supernatant of resting cells, at least one miRNA is differentiallyexpressed (e.g. increased) by statistical significance (p<0.05 formiR-214-3p) and at least one miRNA is modulated by at least a log_(e)fold (e.g. miR-149-5p).

A direct comparison of TA2 miRNA population to IA2 miRNA populations asshown in FIG. 8C, demonstrates that at least two miRNAs aredifferentially expressed by volcano statistical significance (p<0.01 formiR-155-5p and p<0.05 for miR-9-5p) and at least two miRNAs aremodulated by at least a log₂ (e.g. miR-183-5p and mir-147a).

On FIG. 8C, nine miRNA species were identified. These miRNA species wereselected because they were considered to be differentially expressed asdetermined by clustergram analysis between the AI2 and TA2 preparations.The miRNA species identified with 1, 2, 3, 5, 6, 8 and 9 showedincreased abundance in the TA2 preparations relative to the IA2preparations. The miRNA species identified with 4 has a relativeabundance similar in both the IA2 preparation and TA2 preparations andelevated relative to resting cells.

Further characterization of the miRNA population of the IA2 preparationsand the TA2 preparations is provided in fold change analysis. FIG. 9provides a summary of the fold regulation of the purified miRNApreparations differentially expressed in the IA2 preparations and theTA2 preparations when compared to the conditioned medium of restingcells.

FIG. 10 provides a subset of the miRNAs presented in FIG. 9 andexhibiting at least a log₂ fold modulation when compared to restingcells. As indicated in FIG. 10, a subpopulation of miRNAs are decreasedin the TA2 preparations and increased in the IA2 preparations(miR-183-5p, miR-203a, miR363-3p). As also indicated in FIG. 10, anothersubpopulation of miRNAs are increased in the TA2 preparation anddecreased in the AI2 preparations (miR-21-5p, miR-27a-3p, miR 27b-3p,miR-298, miR-34a-5p, let-7a-5p, let-7e-5p, miR-132-3p).

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth, and as follows in the scopeof the appended claims.

REFERENCES

-   Chen A M, Scott M D. Immunocamouflage: prevention of    transfusion-induced graft-versus-host disease via polymer grafting    of donor cells. J Biomed Mater Res A 2003; 67:626-36.-   Chen A M, Scott M D. Comparative analysis of polymer and linker    chemistries on the efficacy of immunocamouflage of murine    leukocytes. Artif Cells Blood Substit Immobil Biotechnol 2006;    34:305-22.-   Murad K L, Gosselin E J, Eaton J W, Scott M D. Stealth cells:    prevention of major histocompatibility complex class II-mediated    T-cell activation by cell surface modification. Blood 1999;    94:2135-41.-   O'Neill D W, Bhardwaj N. Differentiation of peripheral blood    monocytes into dendritic cells. Curr Protoc Immunol; 2005. Chapter    22: Unit 22F.4.Kyluik-Price D. L., Li, L. and Scott, M. D.    Comparative Efficacy of Blood Cell Immunocamouflage by Membrane    Grafting of Methoxypoly(Ethylene Glycol) and Polyethyloxazoline.    Biomaterials 35(1):412-422 (2014).-   Scott M D, Murad K L, Koumpouras F, Talbot M, Eaton J W. Chemical    camouflage of antigenic determinants: stealth erythrocytes. Proc    Natl Acad Sci USA 1997; 94:7566-71.-   Wang D, Toyofuku W M, Chen A M, Scott M D. Induction of    immunotolerance via mPEG grafting to allogeneic leukocytes.    Biomaterials. 2011 December; 32(35):9494-503.

What is claimed is:
 1. A method of modulating a ratio of the level ofregulatory T (Treg) cells to the level of pro-inflammatory T cells in asubject in need thereof, said method comprising administering atherapeutic amount of at least one acellular pro-inflammatorypreparation to the subject having received a therapeutic amount of atleast one acellular pro-tolerogenic preparation, wherein the at leastone acellular pro-tolerogenic preparation is obtained by a first processcomprising: (i) associating a low-immunogenic biocompatible polymer to acytoplasmic membrane of a first leukocyte to obtain a first modifiedleukocyte; (ii) contacting the first modified leukocyte with a secondleukocyte under conditions to allow a pro-tolerogenic allo-recognitionto provide a pro-tolerogenic conditioned preparation, wherein the firstmodified leukocyte is allogeneic to the second leukocyte; (iii) removingthe first modified leukocyte and the second leukocyte from thepro-tolerogenic conditioned preparation under conditions to inhibit RNAdegradation so as to obtain a pro-tolerogenic composition enriched inacellular pro-tolerogenic components; and (iv) formulating thepro-tolerogenic composition of step (iii), under conditions to inhibitRNA degradation, in the acellular pro-tolerogenic preparation foradministration to the subject; and the at least one pro-inflammatorypreparation is obtained by a second process comprising: (a) contacting athird leukocyte with a fourth leukocyte under conditions to allow apro-inflammatory allo-recognition to provide a pro-inflammatoryconditioned preparation, wherein the third leukocyte is allogeneic tothe fourth leukocyte; (b) removing the third leukocyte and the fourthleukocyte from the pro-inflammatory conditioned preparation underconditions to inhibit RNA degradation so as to obtain a pro-inflammatorycomposition enriched in acellular pro-inflammatory components; and (c)formulating the pro-inflammatory composition of step (b), underconditions to inhibit RNA degradation, in the acellular pro-inflammatorypreparation for administration to the subject.
 2. The method of claim 1,further comprising identifying that the subject is need of a decrease ofthe ratio of the level of Treg cells to the level of pro-inflammatory Tcells prior to the administration of the at least one acellularpro-inflammatory preparation.
 3. The method of claim 2, wherein the needfor the decrease of the ratio is for treating, preventing and/oralleviating the symptoms associated with a condition caused orexacerbated by a reduced immune response in the subject.
 4. The methodof claim 3, wherein the condition is a proliferation-associateddisorder.
 5. The method of claim 4, wherein the proliferation-associateddisorder is cancer.
 6. The method of claim 3, wherein the condition isan infection.
 7. The method of claim 6, wherein the infection is atleast one of a parasitic infection, a viral infection, a bacterialinfection and a fungal infection.
 8. The method of claim 3, wherein thecondition is an immune response to a vaccine.
 9. The method of claim 1,further comprising administering to the subject a therapeutic amount ofthe at least one acellular pro-tolerogenic preparation prior to theadministration of the therapeutic amount of the at least one acellularpro-inflammatory preparation.
 10. The method of claim 9, furthercomprising identifying that the subject is need of an increase of theratio of the level of Treg cells to the level of pro-inflammatory Tcells prior to the administration of the at least one acellularpro-tolerogenic preparation.
 11. The method of claim 10, wherein theneed for the increase of the ratio is for treating, preventing and/oralleviating the symptoms associated to an auto-immune disease afflictingthe subject.
 12. The method of claim 11, wherein the auto-immune diseaseis at least one of type I diabetes, rheumatoid arthritis, multiplesclerosis, psoriasis, lupus, immune thrombocytopenia, experimentalautoimmune encephalomyelitis, autoimmune uveitis, inflammatory boweldisease, scleroderma and Crohn's disease.
 13. The method of claim 10,wherein the need for the increase of the ratio is for preventing orlimiting the rejection of transplanted cells or tissue in the subject.14. The method of claim 13, wherein the transplanted cells or tissue areallogeneic or xenogeneic to the subject.
 15. A method of modulating aratio of the level of regulatory T (Treg) cells to the level ofpro-inflammatory T cells in a subject in need thereof, said methodcomprising administering a therapeutic amount of at least one acellularpro-tolerogenic preparation to the subject having received a therapeuticamount of at least one acellular pro-inflammatory preparation, whereinthe at least one acellular pro-tolerogenic preparation is obtained by afirst process comprising: (i) associating a low-immunogenicbiocompatible polymer to a cytoplasmic membrane of a first leukocyte toobtain a first modified leukocyte; (ii) contacting the first modifiedleukocyte with a second leukocyte under conditions to allow apro-tolerogenic allo-recognition to provide a pro-tolerogenicconditioned preparation, wherein the first modified leukocyte isallogeneic to the second leukocyte; (iii) removing the first modifiedleukocyte and the second leukocyte from the pro-tolerogenic conditionedpreparation under conditions to inhibit RNA degradation so as to obtaina pro-tolerogenic composition enriched in acellular pro-tolerogeniccomponents; and (iv) formulating the pro-tolerogenic composition of step(iii), under conditions to inhibit RNA degradation, in the acellularpro-tolerogenic preparation for administration to the subject; and theat least one pro-inflammatory preparation is obtained by a secondprocess comprising: (a) contacting a third leukocyte with a fourthleukocyte under conditions to allow a pro-inflammatory allo-recognitionto provide a pro-inflammatory conditioned preparation, wherein the thirdleukocyte is allogeneic to the fourth leukocyte; (b) removing the thirdleukocyte and the fourth leukocyte from the pro-inflammatory conditionedpreparation under conditions to inhibit RNA degradation so as to obtaina pro-inflammatory composition enriched in acellular pro-inflammatorycomponents; and (c) formulating the pro-inflammatory composition of step(b), under conditions to inhibit RNA degradation, in the acellularpro-inflammatory preparation for administration to the subject.
 16. Themethod of claim 15, further comprising identifying that the subject isneed of an increase of the ratio of the level of Treg cells to the levelof pro-inflammatory T cells prior to the administration of the at leastone acellular pro-tolerogenic preparation.
 17. The method of claim 16,wherein the need for the increase of the ratio is for treating,preventing and/or alleviating the symptoms associated to an auto-immunedisease afflicting the subject.
 18. The method of claim 16, wherein theneed for the increase of the ratio is for preventing or limiting therejection of transplanted cells or tissue in the subject.
 19. The methodof claim 15, further comprising administering to the subject atherapeutic amount of the at least one acellular pro-inflammatorypreparation prior to the administration of the therapeutic amount of theat least one acellular pro-tolerogenic preparation.
 20. The method ofclaim 19, further comprising identifying that the subject is need of adecrease of the ratio of the level of Treg cells to the level ofpro-inflammatory T cells prior to the administration of the at least oneacellular pro-inflammatory preparation.
 21. The method of claim 20,wherein the need for the decrease of the ratio is for treating,preventing and/or alleviating the symptoms associated with a conditioncaused or exacerbated by a reduced immune response in the subject.